The Curcumin Analog EF24 Inhibits Proliferation and Invasion of Triple-Negative Breast Cancer Cells by Targeting the Long Noncoding RNA HCG11/Sp1 Axis.

EF24, a curcumin analog, exerts a potent antitumor effect on various cancers. However, whether EF24 retards the progression of triple-negative breast cancer (TNBC) remains unclear. In this study, we explored the role of EF24 in TNBC and clarified the underlying mechanism. In a mouse model of TNBC xenograft, EF24 administration reduced the tumor volume, suppressed cell proliferation, promoted cell apoptosis, and downregulated long noncoding RNA human leukocyte antigen complex group 11 (HCG11) expression. In TNBC cell lines, EF24 administration reduced cell viability, suppressed cell invasion, and downregulated HCG11 expression. HCG11 overexpression reenhanced the proliferation and invasion of TNBC cell lines suppressed by EF24. The following mechanism research revealed that HCG11 overexpression elevated Sp1 transcription factor (Sp1) expression by reducing its ubiquitination, thereby enhanced Sp1-mediated cell survival and invasion in the TNBC cell line. Finally, the in vivo study showed that HCG11-overexpressed TNBC xenografts exhibited lower responsiveness in response to EF24 treatment. In conclusion, EF24 treatment reduced HCG11 expression, resulting in the degradation of Sp1 expression, thereby inhibiting the proliferation and invasion of TNBC cells.

T-2 toxin upregulates the expression of human cytochrome P450 1A1 (CYP1A1) by enhancing NRF1 and Sp1 interaction.

T-2 toxin is a major pollutant in crops and feedstuffs. Due to its high toxicity in a variety of organisms, T-2 toxin is of great concern as a threat to humans and to animal breeding. Overexpression of CYP1A1 may contribute to carcinogenesis, and CYP1A1 may be a promising target for the prevention and treatment of human malignancies. Therefore, it is essential to understand the regulatory mechanism by which T-2 toxin induces CYP1A1 expression in human cells. In this study, we confirmed that T-2 toxin (100 ng/mL) induced the expression of CYP1A1 in HepG2 cells through NRF1 and Sp1 bound to the promoter instead of through the well-recognized Aromatic hydrocarbon receptors (AhR). In cells treated with T-2 toxin, Sp1, but not NRF1, was significantly upregulated. However, T-2 toxin apparently promoted the interaction between NRF1 and Sp1 proteins, as revealed by IP analysis. Furthermore, in T-2 toxin-treated HepG2 cells, nuclear translocation of NRF1 was enhanced, while knockdown of Sp1 ablated NRF1 nuclear enrichment. Our results revealed that the upregulation of CYP1A1 by T-2 toxin in HepG2 cells depended on enhanced interaction between Sp1 and NRF1. This finding suggests the tumorigenic features of T-2 toxin might be related to the CYP1A1, which provides new insights to understand the toxicological effect of T-2 toxin.

Neuronal Growth Factor regulates Brain Specific Kinase 1 expression by inhibiting promoter methylation and promoting Sp1 recruitment.

Brain specific kinases (BRSKs) are serine/threonine kinases, preferentially expressed in the brain after Embryonic Day 12. Although BRSKs are crucial neuronal development factors and regulation of their enzymatic activity has been widely explored, little is known of their transcriptional regulation. In this work, we show that Neuronal Growth Factor (NGF) increased the expression of Brsk1 in PC12 cells. Furthermore, during neuronal differentiation, Brsk1 mRNA increased through a MAPK-dependent Sp1 activation. To gain further insight into this regulation, we analyzed the transcriptional activity of the Brsk1 promoter in PC12 cells treated with NGF. Initially, we defined the minimal promoter region (-342 to +125 bp) responsive to NGF treatment. This region had multiple Sp1 binding sites, one of which was within a CpG island. In vitro binding assays showed that NGF-induced differentiation increased Sp1 binding to this site and that DNA methylation inhibited Sp1 binding. In vitro methylation of the Brsk1 promoter reduced its transcriptional activity and impaired the NGF effect. To evaluate the participation of DNA methyltransferases in Brsk1 gene regulation, the 5'Aza-dC inhibitor was used. 5'Aza-dC acted synergistically with NGF to promote Brsk1 promoter activity. Accordingly, DNMT3B overexpression abolished the response of the Brsk1 promoter to NGF. Surprisingly, we found Dnmt3b to be a direct target of NGF regulation, via the MAPK pathway. In conclusion, our results provide evidence of a novel mechanism of Brsk1 transcriptional regulation changing the promoter's methylation status, which was incited by the NGF-induced neuronal differentiation process.

Reversal of Sp1 transactivation and TGFß1/SMAD1 signaling by H2S prevent nickel-induced fibroblast activation.

Nickel as a heavy metal is known to bring threat to human health, and nickel exposure is associated with changes in fibroblast activation which may contribute to its fibrotic properties. H2S has recently emerged as an important gasotransmitter involved in numerous cellular signal transduction and pathophysiological responses. Interaction of nickel and H2S on fibroblast cell activation has not been studied so far. Here, we showed that a lower dose of nickel (200 µM) induced the activation of human fibroblast cells, as evidenced by increased cell growth, migration and higher expressions of α-smooth muscle actin (αSMA) and fibronectin, while high dose of nickel (1 mM) inhibited cell viability. Nickel reduced intracellular thiol contents and stimulated oxidative stress. Nickel also repressed the mRNA and protein expression of cystathionine gamma-lyase (CSE, a H2S-generating gene) and blocked the endogenous production of H2S. Exogenously applied NaHS (a H2S donor) had no effect on nickel-induced cell viability but significantly attenuated nickel-stimulated cell migration and the expression of αSMA and fibronectin. In contrast, CSE deficiency worsened nickel-induced αSMA expression. Moreover, H2S incubation reversed nickel-stimulated TGFß1/SMAD1 signal and blocked TGFß1-initiated expressions of αSMA and fibronectin. Nickel inhibited the interaction of Sp1 with CSE promoter but strengthened the binding of Sp1 with TGFß1 promoter, which was reversed by exogenously applied NaHS. These data reveal that H2S protects from nickel-stimulated fibroblast activation and CSE/H2S system can be a potential target for the treatment of tissue fibrosis induced by nickel.

Repair gene O6 -methylguanine-DNA methyltransferase is controlled by SP1 and up-regulated by glucocorticoids, but not by temozolomide and radiation.

Therapy of malignant glioma relies on treatment with the O6 -methylating agent temozolomide (TMZ) concomitant with ionizing radiation followed by adjuvant TMZ. For the treatment of recurrences, DNA chloroethylating drugs are also used. The main killing lesion induced by these drugs is O6 -alkylguanine. Since this damage is repaired by O6 -methylguanine-DNA methyltransferase (MGMT), the repair enzyme represents a most important factor of drug resistance, limiting the therapy of malignant high-grade gliomas. Although MGMT has been shown to be transcriptionally up-regulated in rodents following genotoxic stress, it is still unclear whether human MGMT is subject to up-regulation. Here, we addressed the question whether MGMT in glioma cells is enhanced following alkylating drugs or ionizing radiation, using promoter assays. We also checked the response of glioma cell lines to dexamethasone. In a series of experiments, we found no evidence that the human MGMT promoter is significantly up-regulated following treatment with TMZ, the chloroethylating agent nimustine or radiation. It was activated, however, by dexamethasone. Using deletion constructs, we further show that the basal level of MGMT is mainly determined by the transcription factor SP1. The high amount of SP1 sites in the MGMT promoter likely prevents transcriptional up-regulation following genotoxic stress by neutralizing inducible signals. The regulation of MGMT by miRNAs plays only a minor role, as shown by DICER knockdown experiments. Since high dose dexamethasone concomitant with temozolomide is frequently used in glioblastoma therapy, induction of the MGMT gene through glucocorticoids in MGMT promoter unmethylated cases might cause further elevation of drug resistance, while radiation and alkylating drugs seem not to induce MGMT at transcriptional level.

MicroRNA-124 inhibits the progression of adjuvant-induced arthritis in rats.

OBJECTIVE: MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that act as post-transcriptional regulators. We analysed the in vivo effect of miRNA-124 (miR-124, the rat analogue of human miR-124a) on adjuvant-induced arthritis (AIA) in rats. METHODS: AIA was induced in Lewis rats by injecting incomplete Freund's adjuvant with heat-killed Mycobacterium tuberculosis. Precursor (pre)-miR-124 was injected into the right hind ankle on day 9. Morphological changes in the ankle joint were assessed by micro-CT and histopathology. Cytokine expression was examined by western blotting and real-time RT-PCR. The effect of miR-124 on predicted target messenger RNAs (mRNAs) was examined by luciferase reporter assays. The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase staining. RESULTS: We found that miR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leucocyte infiltration and cartilage or bone destruction. Osteoclast counts and expression level of receptor activator of the nuclear factor κB ligand (RANKL), integrin ß1 (ITGB1) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were reduced in AIA rats treated with pre-miR-124. Luciferase analysis showed that miR-124 directly targeted the 3'UTR of the rat NFATc1, ITGB1, specificity protein 1 and CCAAT/enhancer-binding protein α mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. Both miR-124 and miR-124a directly targeted the 3'-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts. CONCLUSIONS: We found that miR-124 ameliorated AIA by suppressing critical prerequisites for arthritis development, such as RANKL and NFATc1. Thus, miR-124a is a candidate for therapeutic use for human rheumatoid arthritis.

Chemopreventive and chemotherapeutic effect of a novel histone deacetylase inhibitor, by specificity protein 1 in MDA-MB-231 human breast cancer cells.

Histone deacetylase inhibitors (HDACi) have been reported to have potent chemopreventive activity because of their effects on the inhibition of cell growth and apoptosis in human cancer cell lines. In the present study, we investigated the apoptotic effect of a novel HDACi, Ky2, and its molecular mechanism in MDA-MB-231 human breast cancer cells in vitro. The chemopreventive effects of Ky2 in MDA-MB-231 cells were evaluated using the MTS assay, anchorage-independent cell transformation assay, DAPI staining, western blot analysis, reverse transcriptase-PCR, and small interfering RNA. Ky2 enhanced histone acetylation and decreased cell viability. Ky2 induced apoptosis evidenced by nuclear condensation and fragmentation, the accumulation of sub-G1 phase, and caspase-dependent PARP cleavage. In addition, Ky2 released cytochrome c from mitochondria to cytosol through the regulation of mitochondria-related proteins (Bid, Bim, and Bcl-xL). Ky2 markedly decreased the level of Sp1 protein expression through both the decrease of Sp1 mRNA level and proteasome-dependent protein degradation. Interestingly, the apoptotic effect of Ky2 is more potent than SAHA, a well-known HDACi. Furthermore, the knockdown of Sp1 protein by Sp1-specific inhibitor, mithramycin A, and siRNA resulted in the alteration of truncated Bid and Bim to induce apoptosis. Furthermore, Ky2 significantly decreased TPA-induced or EGF-induced neoplastic cell transformation in JB6 cells. Our results suggest that Ky2 may be a potential chemopreventive and chemotherapeutic agent by modulating Sp1 in human breast cancer cells.

Apoptotic effect of dibenzylideneacetone on oral cancer cells via modulation of specificity protein 1 and Bax.

OBJECTIVE: Dibenzylideneacetone (DBA), an analogue of curcumin, has been shown to have potential anticancer effects against several cancers. However, the molecular mechanism underlying anticancer activity of DBA has not been well established yet. In this study, we investigated the function and molecular mechanism of DBA in human oral cancer cells. MATERIALS AND METHODS: The growth-inhibitory and apoptotic effects and related signaling pathways of DBA were evaluated using trypan blue exclusion assay, 4'-6-diamidino-2-phenylindole staining, Western blot analysis, siRNA, and reverse transcription-polymerase chain reaction. RESULTS: DBA inhibited cell growth and induced apoptosis, as evidenced by PARP cleavage, activation of caspase-3, and nuclear condensation. DBA also decreased specificity protein 1 (Sp1) expression through facilitating protein degradation. In addition, DBA enhanced the induction of pro-apoptotic protein Bax, resulting in their conformational change, translocation into mitochondrial outer membrane, and its oligomerization. The down-regulation of Sp1 by siRNA targeting Sp1 and mithramycin A increasingly activated Bax to trigger apoptosis. Moreover, DBA-induced growth inhibition and apoptosis in various human oral cancer cell lines were associated with Sp1 down-regulation and induction of Bax. CONCLUSION: These findings suggest that DBA may be a potential anticancer drug candidate to induce apoptosis through down-regulation of Sp1 in human oral cancer.

Inhibition of transcription factor specificity protein 1 alters the gene expression profile of keratinocytes leading to upregulation of kallikrein-related peptidases and thymic stromal lymphopoietin.

Transcription factor specificity protein 1 (Sp1) is involved in diverse cellular functions. We recently found that Sp1 was significantly decreased in skin biopsy samples obtained from patients with atopic dermatitis (AD) and had an even greater reduction in AD patients with a history of eczema herpeticum. In the current study, we sought to better understand the role of Sp1 in skin biological processes by using a small-interfering RNA (siRNA) technique to knock down Sp1 gene expression in normal human keratinocytes (NHKs) and investigated the genome-wide gene expression profiling of Sp1-silenced NHKs. The gene arrays revealed that 53 genes had greater than 3-fold changes in the expression in Sp1-silenced NHKs as compared with scrambled siRNA-silenced cells. Strikingly, six kallikrein (KLK)-related peptidase genes, namely KLK5, KLK6, KLK7, KLK8, KLK10, and KLK12, were upregulated in NHKs following Sp1 silencing. Functionally, protease activity was significantly enhanced in Sp1-silenced keratinocytes as compared with scrambled siRNA-silenced keratinocytes. Moreover, thymic stromal lymphopoietin (TSLP), an epithelial-derived T(H)2-promoting cytokine, was induced in Sp1-silenced keratinocytes because of elevated KLK activity. These results indicate that Sp1 expression deficiency leads to abnormally increased KLK protease activity in keratinocytes and may contribute to T(H)2 immune responses in the skin by inducing TSLP.

Activation of alpha(1) -adrenergic receptors stimulate the growth of small mouse cholangiocytes via calcium-dependent activation of nuclear factor of activated T cells 2 and specificity protein 1.

UNLABELLED: Small cholangiocytes proliferate via activation of calcium (Ca(2+) )-dependent signaling in response to pathological conditions that trigger the damage of large cyclic adenosine monophosphate-dependent cholangiocytes. Although our previous studies suggest that small cholangiocyte proliferation is regulated by the activation of Ca(2+) -dependent signaling, the intracellular mechanisms regulating small cholangiocyte proliferation are undefined. Therefore, we sought to address the role and mechanisms of action by which phenylephrine, an α(1) -adrenergic agonist stimulating intracellular D-myo-inositol-1,4,5-triphosphate (IP(3) )/Ca(2+) levels, regulates small cholangiocyte proliferation. Small and large bile ducts and cholangiocytes expressed all AR receptor subtypes. Small (but not large) cholangiocytes respond to phenylephrine with increased proliferation via the activation of IP(3) /Ca(2+) -dependent signaling. Phenylephrine stimulated the production of intracellular IP(3) . The Ca(2+) -dependent transcription factors, nuclear factor of activated T cells 2 (NFAT2) and NFAT4, were predominantly expressed by small bile ducts and small cholangiocytes. Phenylephrine stimulated the Ca(2+) -dependent DNA-binding activities of NFAT2, NFAT4, and Sp1 (but not Sp3) and the nuclear translocation of NFAT2 and NFAT4 in small cholangiocytes. To determine the relative roles of NFAT2, NFAT4, or Sp1, we knocked down the expression of these transcription factors with small hairpin RNA. We observed an inhibition of phenylephrine-induced proliferation in small cholangiocytes lacking the expression of NFAT2 or Sp1. Phenylephrine stimulated small cholangiocyte proliferation is regulated by Ca(2+) -dependent activation of NFAT2 and Sp1. CONCLUSION: Selective stimulation of Ca(2+) -dependent small cholangiocyte proliferation may be key to promote the repopulation of the biliary epithelium when large bile ducts are damaged during cholestasis or by toxins.

Apoptotic effect of Polygonum Cuspidatum in oral cancer cells through the regulation of specificity protein 1.

OBJECTIVES: The aim of this study was to evaluate the growth inhibitory and apoptosis-inducing effects and mechanisms of Polygonum cuspidatum root in oral cancer cells. MATERIALS AND METHODS: The testing materials were separated by normal-phase silica gel liquid chromatography. The effect of P. cuspidatum root on apoptotsis and its mechanism were performed using 3-(4,5-dimethylthiazol-20yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) (MTS) assay, western blot analysis, RT-PCR, promoter assay, and (4'-6-Diamidino-2-phenylindole) (DAPI) staining. RESULTS: The methanol extract of P. cuspidatum (MEPC) inhibited the proliferation of oral cancer cells by inducing caspase-dependent apoptosis. Protein and mRNA expression levels and the transactivation of Specificity protein 1 (Sp1) were markedly decreased in KB cells treated with MEPC. Ethyl acetate fraction (EA) from MEPC was more potent than aqueous fraction (AQ) from MEPC to induce apoptosis. F2, F3, and F4 from EA differentially inhibited the growth of KB cells, and it depends on the amount of Emodin in F2, F3, and F4. Moreover, Emodin inhibited oral cancer cell growth and induced caspase-dependent apoptosis by decreasing Sp1. MEPC also decreased an apoptosis-related downstream target of Sp1 protein, survivin. CONCLUSION: The results from this study strongly suggest that MEPC, its fraction, and Emodin may be potential bioactive materials to cause apoptosis mechanism via the down-regulation of Sp1 in oral cancer cells.

Hypoxia and adrenergic function: molecular mechanisms related to Egr-1 and Sp1 activation.

Hypoxia is shown to regulate the stress hormone epinephrine through its biosynthesis by phenylethanolamine N-methyltransferase (PNMT) via PNMT gene activation and transcription factors Egr-1 and Sp1 in adrenal medulla-derived PC12 cells. Moderate hypoxia (5% oxygen) markedly stimulates PNMT promoter-driven luciferase activity in the cells. Hypoxia increases Egr-1 and Sp1 mRNA and nuclear protein content and Egr-1 and Sp1 protein-DNA binding complex formation. Subsequent to transcription factor induction, endogenous PNMT mRNA and protein also increase. Egr-1 and Sp1 binding site inactivation or Egr-1 and Sp1 siRNA inhibit PNMT promoter stimulation by hypoxia. Hypoxia elevates protein kinase A (PKA), phospholipase C (PLC), phosphoinositide 3-kinase, protein kinase C, ERK1/2 mitogen-activated protein kinase and p38 mitogen-activated protein kinase expression while selective inhibitors of these signaling enzymes abrogate hypoxic induction of the PNMT promoter and the rise in Egr-1, Sp1 and PNMT mRNA and protein. PC12 cells lacking PKA or PLCgamma-1 show significant reduction in PNMT promoter activation by hypoxia. Signaling inhibitors do not affect these responses or reduce hypoxic induction of the PNMT promoter to a lesser extent. Findings suggest that Egr-1 and Sp1 through synergistic interaction are critical transcriptional activators for hypoxic stress-regulated adrenergic function controlled via cAMP/PKA and PLC signaling. Identification of Sp1 as a mediator of hypoxia-induced transcriptional activation of PNMT has not been previously been shown. The effects of hypoxia on PNMT and thereby epinephrine may have important ramifications for the stress hormone epinephrine, its ability to regulate behavioral and physiological processes associated with stress and stress-elicited illness.

Chronic prenatal hypoxia induces epigenetic programming of PKC{epsilon} gene repression in rat hearts.

RATIONALE: Epidemiological studies demonstrate a clear association of adverse intrauterine environment with an increased risk of ischemic heart disease in adulthood. Hypoxia is a common stress to the fetus and results in decreased protein kinase C epsilon (PKCepsilon) expression in the heart and increased cardiac vulnerability to ischemia and reperfusion injury in adult offspring in rats. OBJECTIVES: The present study tested the hypothesis that fetal hypoxia-induced methylation of cytosine-phosphate-guanine dinucleotides at the PKCepsilon promoter is repressive and contributes to PKCepsilon gene repression in the heart of adult offspring. METHODS AND RESULTS: Hypoxic treatment of pregnant rats from days 15 to 21 of gestation resulted in significant decreases in PKCepsilon protein and mRNA in fetal hearts. Similar results were obtained in ex vivo hypoxic treatment of isolated fetal hearts and rat embryonic ventricular myocyte cell line H9c2. Increased methylation of PKCepsilon promoter at SP1 binding sites, -346 and -268, were demonstrated in both fetal hearts of maternal hypoxia and H9c2 cells treated with 1% O(2) for 24 hours. Whereas hypoxia had no significant effect on the binding affinity of SP1 to the unmethylated sites in H9c2 cells, hearts of fetuses and adult offspring, methylation of both SP1 sites reduced SP1 binding. The addition of 5-aza-2'-deoxycytidine blocked the hypoxia-induced increase in methylation of both SP1 binding sites and restored PKCepsilon mRNA and protein to the control levels. In hearts of both fetuses and adult offspring, hypoxia-induced methylation of SP1 sites was significantly greater in males than in females, and decreased PKCepsilon mRNA was seen only in males. In fetal hearts, there was significantly higher abundance of estrogen receptor alpha and beta isoforms in females than in males. Both estrogen receptor alpha and beta interacted with the SP1 binding sites in the fetal heart, which may explain the sex differences in SP1 methylation in the fetal heart. Additionally, selective activation of PKCepsilon restored the hypoxia-induced cardiac vulnerability to ischemic injury in offspring. CONCLUSIONS: The findings demonstrate a direct effect of hypoxia on epigenetic modification of DNA methylation and programming of cardiac PKCepsilon gene repression in a sex-dependent manner, linking fetal hypoxia and pathophysiological consequences in the hearts of adult offspring.

Cadmium down-regulation of kidney Sp1 binding to mouse SGLT1 and SGLT2 gene promoters: possible reaction of cadmium with the zinc finger domain of Sp1.

Cadmium (Cd) exposure causes glucosuria (glucose in the urine). Previously, it was shown that Cd exposure of primary cultures of mouse kidney cells (PMKC) decreased mRNA levels of the glucose transporters, SGLT1 and SGLT2 and that Sp1 from Cd-exposed cells displayed reduced binding to the GC boxes of the mouse SGLT1 promoter in vitro. Here, we identified a GC box upstream of mouse SGLT2 gene. ChIP assays on PMKC revealed that exposure to 5 microM Cd abolished Sp1 binding to SGLT1 GC box while it decreased Sp1 binding to SGLT2 GC sequence by 30% in vivo. The in vitro DNA binding assay, EMSA, demonstrated that binding of Sp1 from Cd (7.5 microM)-treated PMKC to the SGLT2 GC probe was 86% lower than in untreated cells. Sp1 is a zinc finger protein. Compared to PMKC exposed to 5 microM Cd alone, inclusion of 5 microM Zn restored SGLT1 and 2 mRNA levels by 15% and 30%, respectively. Cd (10 microM) decreased the binding of recombinant Sp1 (rhSp1) to SGLT1 and SGLT2 GC probes to 12% and 8% of untreated controls. Cd exerted no effect on GC-bound rhSp1. Co-treatment with Cd and Zn showed that added Zn significantly restored rhSp1 binding to the SGLT1 and SGLT2. Addition of Zn post Cd treatment was not stimulatory. We conclude that Cd can replace Zn in Sp1 DNA binding domain to reduce its binding to GC sites in mouse SGLT1 and SGLT2 promoters.

Attenuation of c-Jun and Sp1 expression and p300 recruitment to gene promoter confers the trichostatin A-induced inhibition of 12(S)-lipoxygenase expression in EGF-treated A431 cells.

The mechanism by which the histone deacetylase (HDAC) inhibitor trichostatin A inhibits epidermal growth factor (EGF)-induced human 12(S)-lipoxygenase expression was studied. Trichostatin A treatment of human epidermoid carcinoma A431 cells inhibited the EGF-induced 12(S)-lipoxygenase enzymatic activity in a dose-dependent manner that was consistent with the expression of 12(S)-lipoxygenase mRNA and protein. Confocal microscopy indicated that trichostatin A treatment of cells resulted in downregulation of EGF-induced c-Jun expression. Western blotting revealed that trichostatin A treatment of cells resulted in downregulation of EGF-induced c-Jun and constitutively Sp1 expression. Results of a chromatin immunoprecipitation assay revealed that trichostatin A treatment of cells also upregulated Sp1 acetylation and attenuated the recruitment of Sp1, c-Jun, and p300 to the 12(S)-lipoxygenase gene promoter. These results suggested that trichostatin A inhibited EGF-induced 12(S)-lipoxygenase expression by multiple mechanisms, including the attenuation of c-Jun and Sp1 expression and p300 recruitment to the 12(S)-lipoxygenase gene promoter.

De-repression of the p21 promoter in prostate cancer cells by an isothiocyanate via inhibition of HDACs and c-Myc.

Natural isothiocyanates from cruciferous vegetables have been described as important dietary factors for prostate cancer prevention. Phenethyl isothiocyanate (PEITC), found rich in watercress, induces growth arrest and apoptosis in prostate cancer cells, and also inhibits the testosterone-mediated growth of prostates by regulating the androgen receptor and cell cycle progression in rats. PEITC has been recently identified as an inhibitor of histone deacetylases (HDACs). Herein we describe the mechanism of PEITC-mediated growth attenuation in relation to HDAC inhibition in human prostate cancer cells. Exposure of androgen-dependent prostate cancer cells LNCaP to PEITC resulted in cell cycle arrest and a p53-independent up-regulation of the inhibitors of cyclin-dependent kinases, including p21WAF1 and p27. The mechanism of p21 activation was investigated. PEITC significantly enhanced histone acetylation and induced selective modification of histone methylation for chromatin remodeling. Chromatin immunoprecipitation revealed that the p21 gene was associated with the PEITC-induced hyperacetylated histones. As a result, the chromatin unfolding permitted the transcription activation of the p21 gene. PEITC also significantly reduced the expression of c-Myc which represses p21. Pull-down assays using Sp1 affinity oligo beads of the p21 promoter, showed decreased c-Myc binding to the Sp1 transcriptional complexes in the p21 promoter, resulting in reduced p21 repression. The quantity of PEITC (0.5-1 micro M) effective to mediate cell cycle arrest was less than that for inhibiting c-Myc (2-5 micro M), suggesting that the inhibition of HDACs may be the primary mechanism for p21 activation. The PEITC-mediated growth attenuation of prostate cancer cells includes an interactive mechanism involving HDAC and c-Myc inhibition.

Inhibitory effect of acetyl-11-keto-beta-boswellic acid on androgen receptor by interference of Sp1 binding activity in prostate cancer cells.

Androgen receptor (AR)-mediated signaling is crucial for the development and progression of prostate cancer (PCa). Naturally occurring phytochemicals that target the AR signaling offer significant protection against this disease. Acetyl-11-keto-beta-boswellic acid (AKBA), a compound isolated from the gum-resin of Boswellia carterii, caused G1-phase cell cycle arrest with an induction of p21(WAF1/CIP1), and a reduction of cyclin D1 as well in prostate cancer cells. AKBA-mediated cellular proliferation inhibition was associated with a decrease of AR expression at mRNA and protein levels. Furthermore, the functional biomarkers used in evaluation of AR transactivity showed suppressions of prostate-specific antigen promoter-dependent and androgen responsive element-dependent luciferase activities. Additionally, down-regulation of an AR short promoter mainly containing a Sp1 binding site suggested the essential role of Sp1 for the reduction of AR expression in cells exposed to AKBA. Interruption effect of AKBA on Sp1 binding activity but not Sp1 protein levels was further confirmed by EMSA and transient transfection with a luciferase reporter driven by three copies of the Sp1 binding site of the AR promoter. Therefore, anti-AR properties ascribed to AKBA suggested that AKBA-containing drugs could be used for the development of novel therapeutic chemicals.

Sp1 transcription factor as a target for anthracyclines: effects on gene transcription.

The analysis of how anthracyclines interfere with DNA-protein complexes, and the evaluation of their effects on gene transcription, can promote the development of new more specific anti-tumour agents. Daunorubicin and the bisintercalating anthracycline WP631 (which binds more tightly to DNA) have been compared for their ability to inhibit Sp1-DNA interactions and gene transcription. WP631 is more efficient at inhibiting transcription initiation from promoters containing an Sp1-binding site, and it is a potent inhibitor of Sp1-activated transcription both in vitro and in human cell lines. The analysis of gene expression profiles using arrays, which include several genes containing Sp1-putative binding sites, suggests that changes in the transcriptome induce cell cycle arrest and drive a time-dependent response of cells to death stimuli through distinct pathways, which rely on the anthracycline used and its concentration.

Effect of ammonia on ciliary neurotrophic factor mRNA and protein expression and its upstream signalling pathway in cultured rat astroglial cells: possible implication of c-fos, Sp1 and p38MAPK.

Ciliary neurotrophic factor (CNTF) may be implicated in the pathogenetic mechanisms of hepatic encephalopathy. We tested this hypothesis by treating confluent primary cultures of rat astroglial cells with ammonium chloride for various periods and analysing the effect of ammonia on the signalling pathway that regulates CNTF mRNA and protein expression. Ammonia treatment induced a dose- and time-dependent reduction in CNTF mRNA and protein expression. Surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry analysis of CNTF in the culture medium demonstrated that ammonia also induced a significant decrease in CNTF release. In addition, ammonia affected Sp1 and c-fos, transcription factors that regulate CNTF mRNA and protein expression, which showed partial dephosphorylation and significantly lower mRNA and protein levels. Total content of p38MAPK (for which Sp1 and c-fos are substrates) was unaffected by ammonia, although the diphosphorylated (active) form was significantly reduced after ammonia exposure.

Inhibition of androgen receptor signaling by selenite and methylseleninic acid in prostate cancer cells: two distinct mechanisms of action.

The development of prostate cancer and its progression to a hormone-refractory state is highly dependent on androgen receptor (AR) expression. Recent studies have shown that the selenium-based compound methylseleninic acid (MSeA) can disrupt AR signaling in prostate cancer cells. We have found that selenite can inhibit AR expression and activity in LAPC-4 and LNCaP prostate cancer cells as well but through a different mechanism. On entering the cell, selenite consumes reduced glutathione (GSH) and generates superoxide radicals. Pretreatment with N-acetylcysteine, a GSH precursor, blocked the down-regulation of AR mRNA and protein expression by selenite and restored AR ligand binding and prostate-specific antigen expression to control levels. MSeA reacts with reduced GSH within the cell; however, N-acetylcysteine did not effect MSeA-induced down-regulation of AR and prostate-specific antigen. The superoxide dismutase mimetic MnTMPyP was also found to prevent the decrease in AR expression caused by selenite but not by MSeA. A Sp1-binding site in the AR promoter is a key regulatory component for its expression. Selenite decreased Sp1 expression and activity, whereas MSeA did not. The inhibition of Sp1 by selenite was reversed in the presence of N-acetylcysteine. In conclusion, we have found that selenite and MSeA disrupt AR signaling by distinct mechanisms. The inhibition of AR expression and activity by selenite occurs via a redox mechanism involving GSH, superoxide, and Sp1.