Novel Amiloride Derivatives That Inhibit Bacterial Motility across Multiple Strains and Stator Types.

The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomA/PomB stator complex of Vibrio spp., can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium motility inhibitors have been described since the discovery of phenamil. In this study, we characterized two possible motility inhibitors, HM2-16F and BB2-50F, from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free-swimming bacteria, and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na+ motors comparable to phenamil at matching concentrations and could also stop rotation of tethered cells driven by H+ motors. Bead measurements in the presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na+ and H+ stator types and in pathogenic and nonpathogenic strains. IMPORTANCE Here, we characterized two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. These compounds were shown to inhibit flagellar motility at 10 µM across multiple strains: from nonpathogenic Escherichia coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (enterohemorrhagic E. coli or uropathogenic E. coli [EHEC or UPEC, respectively]), and finally, sodium-powered Vibrio alginolyticus. Broad antimotility compounds such as these are important tools in our efforts to control virulence of pathogens in health and agricultural settings.

The pleiotropic regulation of cyclin D1 by newly identified sesaminol-binding protein ANT2.

The expression of cyclin D1 is upregulated in various cancer cells by diverse mechanisms, such as increases in mRNA levels, the promotion of the translation by mammalian target of rapamycin complex 1 (mTORC1) signaling and the protein stabilization. We here show that sesaminol, a sesame lignan, reduces the expression of cyclin D1 with decreasing mRNA expression levels, inhibiting mTORC1 signaling and promoting proteasomal degradation. We subsequently generated sesaminol-immobilized FG beads to newly identify sesaminol-binding proteins. As a consequence, we found that adenine nucleotide translocase 2 (ANT2), the inner mitochondrial membrane protein, directly bound to sesaminol. Consistent with the effects of sesaminol, the depletion of ANT2 caused a reduction in cyclin D1 with decreases in its mRNA levels, mTORC1 inhibition and the proteasomal degradation of its protein, suggesting that sesaminol negatively regulates the function of ANT2. Furthermore, we screened other ANT2-binding compounds and found that the proliferator-activated receptor-γ agonist troglitazone also reduced cyclin D1 expression in a multifaceted manner, analogous to that of the sesaminol treatment and ANT2 depletion. Therefore, the chemical biology approach using magnetic FG beads employed in the present study revealed that sesaminol bound to ANT2, which may pleiotropically upregulate cyclin D1 expression at the mRNA level and protein level with mTORC1 activation and protein stabilization. These results suggest the potential of ANT2 as a target against cyclin D1-overexpressing cancers.

Role of thioredoxin in the response of normal and transformed cells to histone deacetylase inhibitors.

This study examines the basis of resistance and sensitivity of normal and transformed cells to histone deacetylase inhibitor (HDACi)-induced cell death, specifically the role of caspases and thioredoxin (Trx). An important attribute of HDACis is that they induce cancer cell death at concentrations to which normal cells are relatively resistant, making them well suited for cancer therapy. The mechanism underlying this selectivity has not been understood. In this study we found that the HDACi suberoylanilide hydroxamic acid (SAHA) and MS-275, a benzamide, cause an accumulation of reactive oxygen species (ROS) and caspase activation in transformed but not normal cells. Inhibition of caspases does not block HDACi-induced cell death. These studies provide a possible mechanism that can explain why normal but not certain transformed cells are resistant to HDACi-induced cell death. The HDACi causes an increase in the level of Trx, a major reducing protein for many targets, in normal cells but not in transformed cells. The SAHA-induced increase in Trx activity in normal cells is associated with no increase in ROS accumulation. Transfection of transformed cells with Trx small interfering RNA caused a marked decrease in the level of Trx protein with an increase in ROS, a decrease in cell proliferation, and an increase in sensitivity to SAHA-induced cell death. Thus, Trx, independent of the caspase apoptotic pathway, is an important determinant of resistance of cells to HDACi-induced cell death.

Telomerase activation by histone deacetylase inhibitor in normal cells.

Although telomerase activity is known to be regulated mainly at the level of transcription of the human telomerase catalytic subunit (hTERT) gene, the molecular mechanism underlying tumor-specific expression of telomerase remains unclear. Emerging evidence suggests that reversible acetylation of nucleosomal histones and the resultant changes in the chromatin structure are important processes in gene transcription. In particular, histone deacetylase (HDAC) inhibitors activate the transcription of certain genes by altering the acetylation status of nucleosomal histones. The present study examines the effects of HDAC inhibitor on hTERT gene transcription. Treatment with tricostatin A (TSA) induced significant activation of hTERT mRNA expression and telomerase activity in normal cells, but not in cancer cells. Transient expression assays revealed that TSA activates the hTERT promoter. Furthermore, the proximal 181 bp core promoter of hTERT, which contains two c-Myc and five Sp1 sites, was determined to be the responsible element. Overexpression of Sp1 enhanced responsiveness to TSA, and mutation of Sp1 sites, but not c-Myc sites, of the core promoter of hTERT abrogated this activation. Introduction of the dominant-negative form of the Sp family inhibited TSA activation. These results indicate that HDAC inhibitor activates the hTERT promoter in normal cells, in which Sp1 plays a key role. This finding suggests one way whereby histone deacetylation may be involved in silencing the hTERT gene in normal cells.

Differential activities of 1alpha,25-dihydroxy-16-ene-vitamin D(3) analogs and their 3-epimers on human promyelocytic leukemia (HL-60) cell differentiation and apoptosis.

To clarify physiological role of the carbon 3 (C-3) epimerization of 1alpha,25(OH)(2)D(3) and biologic significance of a 3-epi metabolite of 1alpha,25(OH)(2)D(3), we examined biologic activities of the 3-epimers of 1alpha,25(OH)(2)D(3) and 1alpha,25(OH)(2)-16-ene-D(3) analogs in terms of modulation of cell cycle phase distribution and cell-surface CD11b antigen expression of HL-60 cells, transactivation of vitamin D target genes in transfected cells, stimulation of VDR/RXRalpha heterodimer formation in a rabbit reticulocyte lysates transcription/translation system, stimulation of VDR/RXRalpha/VDRE complex formation, and induction of HL-60 cell apoptosis. The analogs tested here were 1) 1alpha,25(OH)(2)D(3), 2) 1alpha,25(OH)(2)-3-epi-D(3), 3) 1alpha,25(OH)(2)-16-ene-D(3), 4) 1alpha,25(OH)(2)-16-ene-3-epi-D(3), 5) 1alpha,25(OH)(2)-16-ene-23-yne-hexafluoro(F(6))-D(3), 6) 1alpha,25(OH)(2)-16-ene-23-yne-hexafluoro(F(6))-3-epi-D(3), 7) 1alpha,25-(OH)(2)-16-ene-20-epi-23-yne-D(3), and 8) 1alpha,25(OH)(2)-16-ene-20-epi-23-yne-3-epi-D(3). When compared to the 3-natural (beta) analogs, the 3-epi (alpha) analogs were biologically significantly less active. The findings support the hypothesis that the C-3 epimerization is an inactivation pathway of 1alpha,25(OH)(2)D(3) and its analogs in vitamin D target tissues. We also found that the 3-epi analogs, but not the 3-natural (beta) analogs, were the potent inducers of apoptosis of HL-60 cells. These results suggest that the analogs could be divided into two groups, in which the 3-epi analogs were the potent inducers of apoptosis of HL-60 cells, and the 3-natural analogs were the potent modulators of HL-60 cell growth and differentiation. This is the first report demonstrating that the 3-epimerization of the hydroxyl group at C-3 of the A-ring of 1alpha,25(OH)(2)D(3) plays an important role to modulate HL-60 cell differentiation and apoptosis.

Activation of the p21WAF1/CIP1 promoter independent of p53 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through the Sp1 sites.

Suberoylanilide hydroxamic acid (SAHA) is a novel histone deacetylase inhibitor with high potency in inducing differentiation of cultured murine erythroleukemia cells. We have recently demonstrated that SAHA induces cell cycle arrest and apoptosis in human breast cancer cells, accompanied by up-regulation of the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, via a p53-independent mechanism. In this study, we used p21 gene expression as a model system to elucidate the molecular mechanism(s) underlying SAHA-mediated gene activation. Treatment of human breast cancer cell line MCF7 cells with SAHA induced p21 mRNA as a consequence of an immediate-early gene activation. Moreover, SAHA activated the p21 promoter primarily through two Spl sites located at -82 and -69 relative to the transcription start site. Furthermore, Sp1 and Sp3 proteins were the major factors binding to the Spl site of the p21 promoter. However, SAHA did not alter their DNA binding activities, suggesting that SAHA mediates p21 promoter activity by a mechanism other than altering the DNA binding activities of Sp1 and Sp3. Further studies using the GAL4 luciferase assay system demonstrated that both GAL4-Sp1 and GAL4-Sp3 fusion proteins supported SAHA-mediated gene activation from a promoter driven by five GAL4 DNA binding sites, and that GAL4-Sp3 fusion protein was suppressive in the absence of SAHA treatment. Collectively, our results suggest that SAHA activates the p21 promoter through the Spl sites, and that both Spl and Sp3 proteins can mediate SAHA-induced gene activation.

Butyrate as a model for "gene-regulating chemoprevention and chemotherapy.".

Recent progress in molecular genetics has facilitated understanding of the mechanisms of carcinogenesis. However, there is not yet any effective therapy or prevention for cancer based on the molecular mechanisms of carcinogenesis. So-called "gene therapy" for cancer is expected to become a new method of treatment, but there are still several serious problems with gene therapy. As a matter of fact, it seems impossible to adopt gene therapy for prevention. We therefore tried to develop a different method of cancer prevention or therapy based on the molecular mechanisms of carcinogenesis. For instance, the tumor-suppressor gene p53 is mutated in about 50% of human malignancies. It is known that p53 stimulates the promoter activities of p21/WAF1, gadd45 and bax genes, resulting in cell cycle arrest, DNA repair and apoptosis, respectively. Therefore, chemical compounds that can stimulate these genes should compensate for the function of p53. As a model of this, we found that histone deacetylase inhibitors such as butyrate or trichostatin A dramatically stimulate the p21/WAF1 gene promoter through the Spl sites, resulting in cell cycle arrest. Interestingly, another group has recently reported that phenylbutyrate, which is also known as a histone deacetylase inhibitor, is very effective for leukemia patients. We therefore consider methods of up-regulating p21/WAF, gadd45 or bax genes should be useful for cancer therapy and termed this method "Gene-regulating chemotherapy". Theoretically, the chemicals up-regulating such genes should be also useful for chemoprevention, and we also termed it as "Gene-regulating chemoprevention". In conclusion, we propose that "Gene-regulating chemotherapy or chemoprevention" may be a promising new method for cancer therapy or prevention and histone deacetylase inhibitor is a good candidate for this method.

Molecular cloning and functional analysis of the murine bax gene promoter.

Bcl-2-associated X protein (Bax) is a proapoptotic protein and is suggested to have an important role in carcinogenesis. To investigate the mechanism of bax gene transcriptional regulation, we isolated and sequenced the genomic DNA fragment of the 5' flanking region of the murine bax gene, and subcloned its promoter region into a luciferase reporter construction. The murine bax promoter is TATA-less, and the sequence is only partially homologous to that of the human bax promoter. Transient transfection into NIH 3T3 cells using unidirectionally deleted promoters and mutants of Sp1 sites revealed that two Sp1 sites were partially responsible for the basal activity. The murine bax promoter was not responsive to exogenous p53, suggesting that the p53-responsive element may not exist in the region used in our current experiments.

Sp3, but not Sp1, mediates the transcriptional activation of the p21/WAF1/Cip1 gene promoter by histone deacetylase inhibitor.

We previously reported that both sodium butyrate and trichostatin A (TSA), both of which are known as inhibitors of histone deacetylase, arrest human tumor cells at G1 and G2-M and activate the cyclin-dependent kinase inhibitor, the p21/WAF1/Cip1 gene promoter, through the Sp1 sites. In this study, we identified Sp1 and Sp3 as major factors binding to the Sp1 sites of the p21/WAF1/Cip1 promoter in MG63 cells through electrophoretic mobility shift assays and showed that TSA treatment did not change their binding activities. However, GAL4-Sp3 but not GAL4-Sp1 fusion protein supported the TSA-mediated gene induction from a luciferase reporter plasmid driven by five GAL4 DNA-binding sites. Moreover, the ectopic expression of dominant negative Sp3 repressed the enhancement by TSA of the p21/WAF1/Cip1 promoter and Sp1 site-driven promoter. Taken together, these results suggest that histone deacetylase inhibitor up-regulates p21/WAF1/Cip1 transcription by Sp3 but not by Sp1.

Histone deacetylase inhibitor activates the p21/WAF1/Cip1 gene promoter through the Sp1 sites.

Trichostatin A (TSA), a specific histone deacetylase inhibitor, induces histone hyperacetylation and modulates the expression of some genes. We examined the effects of TSA on MG63 cells. TSA induced growth arrest and expression of the p21/WAF1/Cip1 protein. A close correlation between the level of histone acetylation and induction of the p21/WAF1/Cip1 protein was detected. Using several mutant p21/WAF1/Cip1 promoter fragments, mutation of either of two Sp1 sites at -82 or -69 of the p21/WAF1/Cip1 promoter reduced the responsiveness to TSA. This finding indicates that TSA activates the p21/WAF1/Cip1 promoter through the Sp1 sites in a p53-independent manner.

p53-independent induction of WAF1/Cip1 is correlated with osteoblastic differentiation by vitamin D3.

1Alpha,25(OH)2 vitamin D3 (1,25(OH)2D3) can induce differentiation of osteoblastic cells by arresting the cell cycle at G1. The p53-inducible gene, WAF1/Cip1, is one of the inhibitors of cyclin-dependent kinases and can inhibit the phosphorylation of retinoblastoma protein (pRB), thereby keeping pRB functionally active. Here we show that in a p53-null human osteoblastic osteosarcoma MG-63 cell line, 10 nM of 1,25(OH)2D3 completely inhibits cell growth and increases alkaline phosphatase activity, which suggests the induction of osteoblastic differentiation. We also found a p53-independent increase of WAF1/Cip1 mRNA and promoter activation by 1,25(OH)2D3. On the other hand, the expression and the promoter activity of the RB gene decreased after treatment with 1,25(OH)2D3 during the differentiation of MG-63 cells. Our results suggest that the p53-independent WAF1/Cip1 induction by 1,25(OH)2D3 is important for osteoblastic differentiation of MG-63 cells.

Cell cycle-dependent modulation of promoter activities of RB and WAF1/Cip1 genes.

A universal inhibitor of cyclin-dependent kinases, WAF1/Cip1 can dephosphorylate the RB gene product to arrest the cell cycle at the G1 phase. Here we show that the mRNA level and the promoter activities of the RB and WAF1/Cip1 genes exhibit cell cycle-dependent change when cells are released from either serum-starvation or the confluent cell state with serum. RB expression and promoter activity are elevated at middle to late G1. In contrast, the mRNA and promoter activity of the WAF1/Cip1 gene increase at early G1. These results suggest that the RB and WAF1/Cip1 expression and promoter activities depend not only on serum, but also on the cell cycle progression itself. Moreover, we identified the responsive region for serum-released cell cycle progression in the RB promoter and mapped it to the region between -4 and -182 relative to the initiating codon of the RB gene. The region in the WAF1/Cip1 promoter responsible for the serum-released cell cycle progression mapped not to the p53 binding site, but to the 374 base-pair region between -1770 and -1396 from the transcription start site.

Retinoblastoma binding factor 1 site in the core promoter region of the human RB gene is activated by hGABP/E4TF1.

We previously reported two oncogenic point mutations present in the RB (retinoblastoma) gene promoter region, found at consensus Sp1 and ATF sites, respectively, and in two separate hereditary RB families. However, Sp1 protein was shown not to bind to the Sp1 site; this indicated that the Sp1 consensus site mutation was blocking the action of an alternative transcription factor, which we called RBF-1 (retinoblastoma binding factor 1). Subsequent purification of RBF-1 revealed it to be hGABP/E4TF1, a transactivator from the adenovirus early-region 4 promoter. In this study, we directly examined the effects of hGABP/E4TF1 on transactivation of the RB gene promoter through the RBF-1 site. As expected, hGABP/E4TF1 enhanced the core RB promoter activity, whereas it did not stimulate a mutant RBF-1 site. We therefore conclude that the most essential transcription factor in the human RB gene is likely to be hGABP/E4TF1.

Butyrate activates the WAF1/Cip1 gene promoter through Sp1 sites in a p53-negative human colon cancer cell line.

Butyrate is a well known colonic luminal short chain fatty acid, which arrests cell growth and induces differentiation in various cell types. We examined the effect of butyrate on the expression of WAF1/Cip1, a potent inhibitor of cyclin-dependent kinases, and its relation to growth arrest in a p53-mutated human colon cancer cell line WiDr. Five millimolar butyrate completely inhibited the growth of WiDr and caused G1-phase arrest. WAF1/Cip1 mRNA was rapidly induced within 3 h by treatment with 5.0 mM butyrate, and drastic WAF1/Cip1 protein induction was detected. Using several mutant WAF1/Cip1 promoter fragments, we found that the butyrate-responsive elements are two Sp1 sites at -82 and -69 relative to the transcription start site. We also found that a TATA element at -46 and two overlapping consensus Sp1 sites at -60 and -55 are essential for the basal promoter activity of WAF1/Cip1. These findings suggest that butyrate arrests the growth of WiDr by activating the WAF1/Cip1 promoter through specific Sp1 sites in a p53-independent fashion.

Effects of quercetin and/or restraint stress on formation of aberrant crypt foci induced by azoxymethane in rat colons.

The present study examines the effect of dietary quercetin and/or restraint stress on the formation of aberrant crypt foci (ACF) induced by azoxymethane (AOM) in the colon. Female F344 rats were divided into four groups. All animals were given intraperitoneal injections of AOM. The animals were fed a basal diet (group A, C), or a 2% quercetin-supplemented diet (group B, D). The animals were put individually to narrow wire cages for 1 h every day throughout the study to expose them to mild restraint stress (group C, D). At week 5, all the rats were killed and analyzed for ACF in the colon. The number of ACF increased significantly in the animals subjected to stress (p < 0.05). On the other hand, dietary quercetin significantly reduced the number of ACF in both the nonstress (p < 0.001) and stress groups (p < 0.05). These findings suggest that quercetin might have a potential as a chemopreventive drug for colon cancer despite stress.

Histone deacetylase inhibitor activates the WAF1/Cip1 gene promoter through the Sp1 sites.

Treatment of cultured cells with trichostatin A (TSA), a specific histone deacetylase inhibitor, induces the histone hyperacetylation and modulates expression of some mammalian genes. We examined the effects of TSA on cell growth arrest, and its relation to expression of the WAF1/Cip1 gene, a potent inhibitor of cyclin-dependent kinases, in a p53-mutated human osteosarcoma cell line MG63. TSA at 500 ng/ml induced growth arrest at both G1 and G2/M phases, and the expressions of the WAF1/Cip1 mRNA and protein. We also examined the changes of acetylated isoforms of histone H4. Dose-response and kinetic analysis suggest a close correlation between the level of histone acetylation and the induction of the WAF1/Cip1 expressions. Using several mutant WAF1/Cip1 promoter fragments, we found that the TSA responsive elements are two Sp1 sites at -82 and -69 relative to the transcription start site. These findings indicate that TSA induces the WAF1/Cip1 promoter through the typical Sp1 sites, in a p53-independent fashion. Furthermore, the Sp1-luc plasmid, containing SV40 promoter-derived three consensus Sp1 binding sites, was markedly activated by TSA, compared to the mutant Sp1-luc plasmid. These results demonstrate that transcriptional activation through the Sp1 sites of the WAF1/Cip1 promoter by TSA coincides with induced hyperacetylation of histone H4.

ATF site of human RB gene promoter is a responsive element of myogenic differentiation.

RB mRNA increases during terminal differentiation of C2 myoblasts. We demonstrate that RB promoter activity increases about 4-fold during differentiation. The increase of RB promoter activity was reduced when a point mutation was designed in the ATF site. In a gel shift assay of the ATF site, two specific bands were observed. One of them, with the lower mobility, disappeared during differentiation. This band reacted with an antibody against ATF-1. We cotransfected an RB promoter-luciferase plasmid with the TREB36/ATF-1 plasmid. ATF-l suppressed the activity of the wild-type RB promoter but not of that with a point mutation at the ATF site. These results suggest that the ATF site of the RB promoter is a responsive element during myogenic differentiation of C2 cells. We hypothesize that RB promoter activity is stimulated partially due to the dissociation of ATF-1, which suppresses the promoter activity through the ATF site in C2 myoblasts.

The retinoblastoma binding factor 1 (RBF-1) site in RB gene promoter binds preferentially E4TF1, a member of the Ets transcription factors family.

The tumor suppressor retinoblastoma gene product, pRB, is a well known regulator of G1/S cell cycle progression. Moreover, mutational inactivations within the retinoblastoma gene (RB) are found in many human malignant tumors, and thus, believed to be an essential step in tumor formation. The human RB gene is considered as a housekeeping gene with no characteristic TATA or CAAT elements in its promoter region, but the sequence between 206 and 185 bases upstream of the initiation codon, essential for RB promoter activity, contains putative Sp1 and ATF recognition sites. We have previously reported that point mutations in this region, causing low penetrance retinoblastomas, completely reduced RB promoter activity, and that a nuclear factor, named RBF-1 (retinoblastoma binding factor 1), could specifically bind to this sequence, overlapping Sp1 recognition sequence. We show here, that RBF-1 can recognize a specific DNA sequence, 5'-GGCGGAAGT-3', overlapping the Sp1 and ATF sites and corresponding to the consensus DNA binding site for members of Ets transcription factors family. When RBF-1 site was used for sequence specific DNA affinity purification from erythroleukemia cells, reconstitution assays, immunoblotting analysis and peptide mapping show that the two major co-purified proteins are identical to human E4TF1-60 and -53 proteins. This reveals that E4TF1 can bind to the RBF-1 site of RB gene promoter, which, thus, constitutes a new target for this member of Ets transcription factors family.