Role of nuclear factor kappa B and reactive oxygen species in the tumor necrosis factor-a-induced epithelial-mesenchymal transition of MCF-7 cells

The microenvironment of the tumor plays an important role in facilitating cancer progression and activating dormant cancer cells. Most tumors are infiltrated with inflammatory cells which secrete cytokines such as tumor necrosis factor-a (TNF-a). To evaluate the role of TNF-a in the development of cancer we studied its effects on cell migration with a migration assay. The migrating cell number in TNF-a -treated group is about 2-fold of that of the control group. Accordingly, the expression of E-cadherin was decreased and the expression of vimentin was increased upon TNF-a treatment. These results showed that TNF-a can promote epithelial-mesenchymal transition (EMT) of MCF-7 cells. Further, we found that the expression of Snail, an important transcription factor in EMT, was increased in this process, which is inhibited by the nuclear factor kappa B (NFkB) inhibitor aspirin while not affected by the reactive oxygen species (ROS) scavenger N-acetyl cysteine. Consistently, specific inhibition of NFkB by the mutant IkBa also blocked the TNF-a-induced upregulation of Snail promoter activity. Thus, the activation of NFkB, which causes an increase in the expression of the transcription factor Snail is essential in the TNF-a-induced EMT. ROS caused by TNF-a seemed to play a minor role in the TNF-a-induced EMT of MCF-7 cells, though ROS per se can promote EMT. These findings suggest that different mechanisms might be responsible for TNF-a - and ROS-induced EMT, indicating the need for different strategies for the prevention of tumor metastasis induced by different stimuli.

Transcriptional up-regulation of PHLDA1 by 17β-estradiol in MCF-7 breast cancer cells

Most breast cancer risk factors are associated with prolonged exposure of the mammary gland to high levels of estrogens. The actions of estrogens are predominantly mediated by two receptors, ERα and ERβ, which act as transcription factors binding with high affinity to estrogen response elements in the promoter region of target genes. However, most target genes do not contain the consensus estrogen response elements, but rather degenerated palindromic sequences showing one or more mutations and other ER-binding sites such as AP-1 and SP-1. Using the differential display reverse transcription-polymerase chain reaction technique, our group identified several genes differentially expressed in normal tissue and in ER-positive and ER-negative primary breast tumors. One of the genes shown to be down-regulated in breast tumors compared to normal breast tissue was the PHLDA1 (Pleckstrin homology-like domain, family A, member 1). In the present study, we investigated the potential of PHLDA1 to be regulated by estrogen via ER in MCF-7 breast cancer cells. The promoter region of PHLDA1 shows an imperfect palindrome, an AP-1- and three SP-1-binding sites potentially regulated by estrogens. We also assessed the effects of 17β-estradiol on PHLDA1 mRNA expression in MCF-7 breast cancer cells. MCF-7 cells exposed to 10 nM 17β-estradiol showed more than 2-fold increased expression of the PHLDA1 transcripts compared to control cells (P = 0.05). The anti-estrogen ICI 182,780 (1 µM) inhibited PHLDA1 mRNA expression and completely abolished the effect of 10 nM 17β-estradiol on PHLDA1 expression (P < 0.05), suggesting that PHLDA1 is regulated by estrogen via ER.

Efficacy of the dietary histone deacetylase inhibitor butyrate alone or in combination with vitamin A against proliferation of MCF-7 human breast cancer cells

The combined treatment with histone deacetylase inhibitors (HDACi) and retinoids has been suggested as a potential epigenetic strategy for the control of cancer. In the present study, we investigated the effects of treatment with butyrate, a dietary HDACi, combined with vitamin A on MCF-7 human breast cancer cells. Cell proliferation was evaluated by the crystal violet staining method. MCF-7 cells were plated at 5 x 10(4) cells/mL and treated with butyrate (1 mM) alone or combined with vitamin A (10 µM) for 24 to 120 h. Cell proliferation inhibition was 34, 10 and 46% following treatment with butyrate, vitamin A and their combination, respectively, suggesting that vitamin A potentiated the inhibitory activities of butyrate. Furthermore, exposure to this short-chain fatty acid increased the level of histone H3K9 acetylation by 9.5-fold (Western blot), but not of H4K16, and increased the expression levels of p21WAF1 by 2.7-fold (Western blot) and of RARβ by 2.0-fold (quantitative real-time PCR). Our data show that RARβ may represent a molecular target for butyrate in breast cancer cells. Due to its effectiveness as a dietary HDACi, butyrate should be considered for use in combinatorial strategies with more active retinoids, especially in breast cancers in which RARβ is epigenetically altered.

Synergistic antitumor effect of TRAIL and adriamycin on the human breast cancer cell line MCF-7

The aim of the present study was to determine the effect of the combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and adriamycin (ADM) on the human breast cancer cell line MCF-7 and to identify potential mechanisms of apoptosis. Cell viability was analyzed by the MTT assay and the synergistic effect was assessed by the Webb coefficient. Apoptosis was quantified using the annexin V-FITC and propidium iodide staining flow cytometry. The mRNA expression of TRAIL receptors was measured by RT-PCR. Changes in the quantities of Bax and caspase-9 proteins were determined by Western blot. MCF-7 cells were relatively resistant to TRAIL (IC50 >10 µg/mL), while MCF-7 cells were sensitive to ADM (IC50 <10 µg/mL). A subtoxic concentration of ADM (0.5 µg/mL) combined with 0.1, 1, or 10 µg/mL TRAIL had a synergistic cytotoxic effect on MCF-7 cells, which was more marked with the combination of TRAIL (0.1 µg/mL) and ADM (0.5 µg/mL). In addition, the combined treatment with TRAIL and ADM significantly increased cell apoptosis from 9.8% (TRAIL) or 17% (ADM) to 38.7%, resulting in a synergistic apoptotic effect, which is proposed to be mediated by up-regulation of DR4 and DR5 mRNA expression and increased expression of Bax and caspase-9 proteins. These results suggest that the combination of TRAIL and ADM might be a promising therapy for breast cancer.

Transcriptional regulation of bidirectional gene pairs by 17-β-estradiol in MCF-7 breast cancer cells

Using cDNA microarray analysis, we previously identified a set of differentially expressed genes in primary breast tumors based on the status of estrogen and progesterone receptors. In the present study, we performed an integrated computer-assisted and manual search of potential estrogen response element (ERE) binding sites in the promoter region of these genes to characterize their potential to be regulated by estrogen receptors (ER). Publicly available databases were used to annotate the position of these genes in the genome and to extract a 5’flanking region 2 kb upstream to 2 kb downstream of the transcription start site for transcription binding site analysis. The search for EREs and other binding sites was performed using several publicly available programs. Overall, approximately 40% of the genes analyzed were potentially able to be regulated by estrogen via ER. In addition, 17% of these genes are located very close to other genes organized in a head-to-head orientation with less than 1.0 kb between their transcript units, sharing a bidirectional promoter, and could be classified as bidirectional gene pairs. Using quantitative real-time PCR, we further investigated the effects of 17β-estradiol and antiestrogens on the expression of the bidirectional gene pairs in MCF-7 breast cancer cells. Our results showed that some of these gene pairs, such as TXNDC9/EIF5B, GALNS/TRAPPC2L, and SERINC1/PKIB, are modulated by 17β-estradiol via ER in MCF-7 breast cancer cells. Here, we also characterize the promoter region of potential ER-regulated genes and provide new information on the transcriptional regulation of bidirectional gene pairs.

Co-culture of apoptotic breast cancer cells with immature dendritic cells: a novel approach for DC-based vaccination in breast cancer

A dendritic cell (DC)-based vaccine strategy could reduce the risk of recurrence and improve the survival of breast cancer patients. However, while therapy-induced apoptosis of hepatocellular and colorectal carcinoma cells can enhance maturation and antigen presentation of DCs, whether this effect occurs in breast cancer is currently unknown. In the present study, we investigated the effect of doxorubicin (ADM)-induced apoptotic MCF-7 breast cancer cells on the activation of DCs. ADM-induced apoptotic MCF-7 cells could effectively induce immature DC (iDC) maturation. The mean fluorescence intensity (MFI) of DC maturity marker CD83 was 23.3 in the ADM-induced apoptotic MCF-7 cell group compared with 8.5 in the MCF-7 cell group. The MFI of DC co-stimulatory marker CD86 and HLA-DR were also increased after iDCs were treated with ADM-induced apoptotic MCF-7 cells. Furthermore, the proliferating autologous T-lymphocytes increased from 14.2 to 40.3% after incubated with DCs induced by apoptotic MCF-7 cells. The secretion of interferon-γ by these T-lymphocytes was also increased. In addition, cell-cell interaction between apoptotic MCF-7 cells and iDCs, but not soluble factors released by apoptotic MCF-7 cells, was crucial for the maturation of iDCs. These findings constitute a novel in vitro DC-based vaccine strategy for the treatment of breast cancer by ADM-induced apoptotic MCF-7 cells.

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

RNAi-mediated knockdown of FANCF suppresses cell proliferation, migration, invasion, and drug resistance potential of breast cancer cells

Fanconi anemia complementation group F protein (FANCF) is a key factor, which maintains the function of FA/BRCA, a DNA damage response pathway. However, the functional role of FANCF in breast cancer has not been elucidated. We performed a specific FANCF-shRNA knockdown of endogenous FANCF in vitro. Cell viability was measured with a CCK-8 assay. DNA damage was assessed with an alkaline comet assay. Apoptosis, cell cycle, and drug accumulation were measured by flow cytometry. The expression levels of protein were determined by Western blot using specific antibodies. Based on these results, we used cell migration and invasion assays to demonstrate a crucial role for FANCF in those processes. FANCF shRNA effectively inhibited expression of FANCF. We found that proliferation of FANCF knockdown breast cancer cells (MCF-7 and MDA-MB-435S) was significantly inhibited, with cell cycle arrest in the S phase, induction of apoptosis, and DNA fragmentation. Inhibition of FANCF also resulted in decreased cell migration and invasion. In addition, FANCF knockdown enhanced sensitivity to doxorubicin in breast cancer cells. These results suggest that FANCF may be a potential target for molecular, therapeutic intervention in breast cancer.

Involvement of miR-30c in resistance to doxorubicin by regulating YWHAZ in breast cancer cells

MicroRNAs (miRNAs) are small RNA molecules that modulate gene expression implicated in cancer, which play crucial roles in diverse biological processes, such as development, differentiation, apoptosis, and proliferation. The aim of this study was to investigate whether miR-30c mediated the resistance of breast cancer cells to the chemotherapeutic agent doxorubicin (ADR) by targeting tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ). miR-30c was downregulated in the doxorubicin-resistant human breast cancer cell lines MCF-7/ADR and MDA-MB-231/ADR compared with their parental MCF-7 and MDA-MB-231 cell lines, respectively. Furthermore, we observed that transfection of an miR-30c mimic significantly suppressed the ability of MCF-7/ADR to resist doxorubicin. Moreover, the anti-apoptotic gene YWHAZ was confirmed as a target of miR-30c by luciferase reporter assay, and further studies indicated that the mechanism for miR-30c on the sensitivity of breast cancer cells involved YWHAZ and its downstream p38 mitogen-activated protein kinase (p38MAPK) pathway. Together, our findings provided evidence that miR-30c was one of the important miRNAs in doxorubicin resistance by regulating YWHAZ in the breast cancer cell line MCF-7/ADR.

Produtos naturais da ascídia Botrylloides giganteum, das esponjas Verongula gigantea, Ircinia felix, Cliona delitrix e do nudibrânquio Tambja eliora, da costa do Brasil

Two new marine metabolites, 3Z, 6Z, 9Z-dodecatrien-1-ol (1) from the ascidian Botrylloides giganteum and 4H-pyran-2ol acetate from the sponge Ircinia felix (4) are herein reported. The known bromotyrosine compounds, 2-(3,5-dibromo-4-methoxyphenyl)-N,N,N-dimethylethanammonium (2) and 2,6-dibromo-4-(2-(trimethylammonium)ethyl)phenol (3), have been isolated from the sponge Verongula gigantea. Serotonin (5) is reported for the first time from the sponge Cliona delitrix, and tambjamines A (15) and D (16) isolated as their respective salts from the nudibranch Tambja eliora. Only tambjamine D presented cytotoxicity against CEM (IC50 12.2 µg/mL) and HL60 (IC50 13.2 µg/mL) human leukemya cells, MCF-7 breast cancer cells (IC50 13.2 µg/mL), colon HCT-8 cancer cells (IC50 10.1 µg/mL) and murine melanoma B16 cancer cells (IC50 6.7 µg/mL).

Synthesis of geranylhydroquinone derivatives with potencial cytotoxic activity

Natural geranylhydroquinone 1 and geranyl-p-methoxyphenol 2 were prepared by Electrophilic Aromatic Substitution (EAS) reactions between geraniol and 1,4-hydroquinone or p-methoxyphenol respectively, using BF3∙Et2O as a catalyst. Furthermore, natural geranylquinone 3, geranyl-1,4-dimethoxyquinone 4 and the new geranyl-4-methoxyphenyl acetate 5 were obtained by chemical transformations of 1 and 2. The compounds were evaluated for their in vitro cytotoxicity activities against cultured human cancer cells of PC-3 human prostate cancer, MCF-7 and MDA-MB-231 breast carcinoma, and Dermal Human Fibroblasts DHF. IC50 values were in the µM range.

Estudo químico de Sinningia allagophylla guiado por testes de atividade antiproliferativa

Activity guided fractionation of Sinningia allagophylla (Mart.) Wiehler ethanolic extract yielded a new benzochromene 8-methoxylapachenol, besides seven known compounds: lapachenol, sitosteryl oleate, sitosteryl linoleate, stigmasteryl oleate, stigmasteryl linoleate, dunniol and tectoquinone. Extract, fractions, and compounds lapachenol, 8-methoxylapachenol, and dunniol were tested in vitro against human cancer cell lines U251 (glioma, CNS), MCF-7 (breast), NCI-ADR/RES (drug-resistant ovarian), 786-0 (kidney), NCI-H460 (lung, no small cells), PC-3 (prostate), OVCAR-3 (ovarian), HT-29 (colon), K562 (leukemia) and against VERO, a normal cell line. The most active compound was dunniol, which inhibited the growth of U251, MCF-7, NCI-ADR/RES, OVCAR-3 and K562 cell lines.

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

Flucytosine + fluconazole association in the treatment of a murine experimental model of cryptococcosis

The efficacy of flucytosine (5-FC) and fluconazole (FLU) association in the treatment of a murine experimental model of cryptococcosis, was evaluated. Seven groups of 10 Balb C mice each, were intraperitoneally inoculated with 10(7) cells of Cryptococcus neoformans. Six groups were allocated to receive 5-FC (300 mg/kg) and FLU (16 mg/ kg), either combined and individually, by daily gavage beginning 5 days after the infection, for 2 and 4 weeks. One group received distilled water and was used as control. The evaluation of treatments was based on: survival time; macroscopic examination of brain, lungs, liver and spleen at autopsy; presence of capsulated yeasts in microscopic examination of wet preparations of these organs and cultures of brain homogenate. 5-FC and FLU, individually or combined, significantly prolonged the survival time of the treated animals with respect to the control group (p<0.01). Animals treated for 4 weeks survived significantly longer than those treated for 2 weeks (p<0.01). No significant differences between the animals treated with 5-FC and FLU combined or separately were observed in the survival time and morphological parameters. The association of 5-FC and FLU does not seem to be more effective than 5-FC or FLU alone, in the treatment of this experimental model of cryptococcosis.

Blood culture as a parameter of treatment effectiveness in experimental histoplasmosis of the hamster

The aim of this study was to determine the value of blood culture as a parameter of treatment effectiveness in experimental histoplasmosis. A total of thirty five hamsters, weighing approximately 120g, were inoculated intracardiacly with 0.1 ml of a suspension containing 4 x 10(7) cells/ml of the yeast phase of H. capsulatum. Treatments were started one week after the infection and lasted for 3 weeks. The azoles, (itraconazole, saperconazole and fluconazole) were administered once a day by gavage, at a dose of 8 mg/kg; Amphotericin B was given intraperitonealy every other day at a dose of 6mg/kg. Blood samples (1 ml) were obtained by heart punction from the 4th day after infection and were seeded in Sabouraud honey-agar and BHI-agar. The hamsters that survived were killed one week after treatment completion and the following criteria were considered for treatment evaluation: 1) rate of spontaneous death, at the end of the experience; 2) microscopic examination of Giemsa smears from liver and spleen and 3) determination of CFU in spleen cultures. Amphotericin B was the most effective drug, with negative blood cultures at day 20, negative spleen cultures in all cases and all the animals survived until the end of the study. Fluconazole was the less effective drug, blood cultures were positive during the whole experience, spleen cultures showed a similar average of CFU when compared with the control animals and 42.8% of these animals died. Saperconazole and itraconazole showed a similar activity, with survival of all hamsters and negative blood cultures at 23 and 26 days respectively. Blood culture seems to be valuable parameter for treatments' evaluation in experimental histoplasmosis of the hamster.