Synthesis and antibacterial activity of novel lincomycin derivatives. III. Optimization of a phenyl thiadiazole moiety.

Lincomycin derivatives that have a 5-(2-nitrophenyl)-1,3,4-thiadiazol-2-yl thio moiety at the 7-position were synthesized. 5-Substituted 2-nitrophenyl derivatives showed potent antibacterial activities against Streptococcus pneumoniae and Streptococcus pyogenes with erm gene. Antibacterial activities of the 4,5-di-substituted 2-nitrophenyl derivatives were generally comparable to those of telithromycin (TEL) against S. pneumoniae with erm gene and clearly superior to those of TEL against S. pyogenes with erm gene. Compounds 6 and 10c that have a methoxy group at the 5-position of the benzene ring exhibited activities comparable to TEL against Haemophilus influenzae. These results suggest that lincomycin derivatives modified at the 7-position would be promising compounds as a clinical candidate. We would like to dedicate this article to the special issue for late Professor Dr. Hamao Umezawa in The Journal of Antibiotics.The Journal of Antibiotics advance online publication, 5 July 2017; doi:10.1038/ja.2017.59.

Synthesis and structure-activity relationships of novel lincomycin derivatives. Part 2. Synthesis of 7(S)-7-deoxy-7-(4-morpholinocarbonylphenylthio)lincomycin and its 3-dimensional analysis with rRNA.

Lincomycin derivatives, which possess a hetero ring at the C-7 position via sulfur atom, were synthesized by three types of reactions: (1) Mitsunobu reaction of 2,3,4-tris-O-(trimethylsiliyl)lincomycin (1) with the corresponding thiol, (2) SN2 reaction of 7-O-methanesulfonyl-2,3,4-tris-O-(trimethylsiliyl)lincomycin (2) with the corresponding thiol and (3) Pd-catalyzed cross-coupling reaction of 7-deoxy-7-epi-7-mercaptolincomycin (35) with the corresponding aryl halides. As a result, compound 28 had potent antibacterial activities against major pathogens, which caused respiratory infections, even compared with clindamycin. On the other hand, compound 38 showed most potent activities against a variety of Streptococcus pneumoniae with erm gene.

The knockdown of endogenous replication factor C4 decreases the growth and enhances the chemosensitivity of hepatocellular carcinoma cells.

AIMS: To identify differentially expressed genes and thereby detect potential molecular targets for future therapies directed against hepatocellular carcinoma (HCC). METHODS: To isolate differentially expressed genes between HCC and adjacent non-cancerous liver tissues, cDNA microarray and quantitative reverse transcriptase polymerase chain reaction analyses were performed. Gene knockdown experiments in HepG2 cells were also performed using small interfering RNAs (siRNAs). Proteins were detected by immunostaining, and cell proliferation was analysed using the MTT/WST-8 assay. Apoptosis and cell cycle analyses were performed using flow cytometry. RESULTS: After an intensive screening for differentially expressed genes in HCC tissues, we isolated 23 upregulated genes in these lesions. Among these, we focused on the replication factor C4 (RFC4) gene. The expression of endogenous RFC4 proteins in HepG2 cells was found to be significantly reduced by RFC4-specific siRNA. This inhibition of RFC4 expression correlated with a decrease in cellular proliferation, increased levels of apoptosis and a sensitizing of the cells to the DNA-damaging chemotherapeutic agents, doxorubicin and camptothecin. CONCLUSION: The replication factor C4 gene may be a novel target for developing cancer therapeutics, which can enhance the antitumour activity of chemotherapeutic agents that induce DNA damage.

Carbonyl Reductase 3 (CBR3) Mediates 9-cis-Retinoic Acid-Induced Cytostatis and is a Potential Prognostic Marker for Oral Malignancy.

The molecular mechanisms of growth suppression by retinoic acid (RA) were examined. Our results suggest that the cytostatic effects of RA could be mediated by the activation of endogenous CBR3 gene in oral squamous cell carcinomas (OSCCs), and the expression is a potential marker for oral malignancy.

Expression of centromere protein F (CENP-F) associated with higher FDG uptake on PET/CT, detected by cDNA microarray, predicts high-risk patients with primary breast cancer.

BACKGROUND: Higher standardized uptake value (SUV) detected by 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) correlates with proliferation of primary breast cancer. The purpose of this study is to identify specific molecules upregulated in primary breast cancers with a high SUV and to examine their clinical significance. METHODS: We compared mRNA expression profiles between 14 tumors with low SUVs and 24 tumors with high SUVs by cDNA microarray. We identified centromere protein F (CENP-F) and CDC6 were upregulated in tumors with high SUVs. RT-PCR and immunohistochemical analyses were performed to validate these data. Clinical implication of CENP-F and CDC6 was examined for 253 archival breast cancers by the tissue microarray. RESULTS: The relative ratios of CENP-F and CDC6 expression levels to beta-actin were confirmed to be significantly higher in high SUV tumors than in low SUV tumors (p = 0.027 and 0.025, respectively) by RT-PCR. In immunohistochemical analysis of 47 node-negative tumors, the CENP-F expression was significantly higher in the high SUV tumors (74%) than the low SUV tumors (45%) (p = 0.04), but membranous and cytoplasmic CDC6 expressions did not significantly differ between both groups (p = 0.9 each). By the tissue microarray, CENP-F (HR = 2.94) as well as tumor size (HR = 4.49), nodal positivity (HR = 4.1), and Ki67 (HR = 2.05) showed independent impact on the patients' prognosis. CONCLUSION: High CENP-F expression, correlated with high SUV, was the prognostic indicators of primary breast cancer. Tumoral SUV levels may serve as a pretherapeutic indicator of aggressiveness of breast cancer.

Gene expression signatures that classify the mode of invasion of primary oral squamous cell carcinomas.

To identify molecular signatures and establish a new diagnostic model for progressive oral squamous cell carcinoma (OSCC). Total RNAs were isolated from primary OSCCs from both node-positive and -negative patients and used in cDNA microarray analysis. To identify marker genes representing a malignant phenotype, their expression was further examined by quantitative reverse transcription-PCR (QRT-PCR) in 64 OSCC tissues. Using Fisher's linear discriminant analysis (LDA) fitted with a stepwise increment method, we created discriminatory predictor models. The stability of these models was examined using leave-one-out cross validation. Immunohistochemical analysis was performed. Among the 16,600 possible target cDNAs in the array analysis, 83 genes demonstrated significantly differential signals (>2-fold). We further identified 53 marker genes that can be implicated in the Yamamoto-Kohama's (YKs) mode of invasion for OSCCs (P < 0.06). Using LDA fitted with a stepwise increment method, we created four discriminatory predictor models based on 16- to 25-gene signatures which could best distinguish the five established grades of YKs mode of invasion. Leave-one out validation demonstrated that the stability of these models was 92-95%. For validation, we also examined an independent set of 13 primary OSCCs; the predictor models determined the invasion status from 77% to 100% (on average, 85%) fidelity with the pathological observations. TGM3 protein expression was markedly suppressed in highly invasive OSCCs. We reveal novel gene expression alterations during the progression of OSCC, and have constructed prediction models for the evaluation of the invasion status of these cancers.

Clinicopathological and prognostic relevance of uptake level using 18F-fluorodeoxyglucose positron emission tomography/computed tomography fusion imaging (18F-FDG PET/CT) in primary breast cancer.

OBJECTIVE: Using integrated 18F-fluorodeoxyglucose positron emission tomography/computed tomography fusion imaging (18F-FDG PET/CT), the clinical significance of 18F-FDG uptake was evaluated in patients with primary breast cancer. METHODS: Clinicopathological correlation with the level of maximum standardized uptake values (SUV) 60 min obtained from preoperative 18F-FDG PET/CT were examined in 152 patients with primary breast cancer. The prognostic impact of the level of SUV was explored using simulated prognosis derived from computed program Adjuvant! in 136 (89%) patients with invasive ductal carcinoma (IDC). RESULTS: High SUV level was significantly correlated with tumor invasive size (< or = 2 cm) (P < 0.0001), higher score of nuclear grade (P < 0.0001), nuclear atypia (P < 0.0001) and mitosis counts (P < 0.0001), negative hormone receptor status (P = 0.001), high score of c-erbB-2 expression (P = 0.006), lymph node metastasis (P = 0.002), and IDC in comparison with invasive lobular carcinoma (P = 0.004). Multivariate analyses showed tumor invasive size, nuclear grade and estrogen receptor negativity were significantly correlated with SUV in primary breast cancer (P < 0.0001,< 0.0001, and < 0.012, respectively), and nuclear grade was significantly correlated with SUV in tumors of invasive size 2 cm or less (P < 0.0001). Tumors with high SUV (cutoff value 4.0) showed higher relapse and mortality rate compared to those with low SUV (P < 0.0001). CONCLUSIONS: High uptake of 18F-FDG would be predictive of poor prognosis in patients with primary breast cancer, and aggressive features of cancer cells in patients with early breast cancer. 18F-FDG PET/CT could be a useful tool to pre-therapeutically predict biological characteristics and baseline risk of breast cancer.

BIGH3 is overexpressed in clear cell renal cell carcinoma.

To identify new target marker genes in renal cell carcinoma (RCC), we compared the gene expression profiles of clear cell RCC (cc-RCC) and normal kidney tissue using serial analysis of gene expression. Our results revealed that the transforming growth factor beta induced 68 kDa protein (TGF-betaI: beta ig-h3 (BIGH3), plasminogen activator inhibitor-1 (PAI-1) and transforming growth factor beta1 (TGF-beta1) genes are up-regulated in cc-RCC. To further assess the role of BIGH3 in RCC, we investigated the mRNA expression levels of BIGH3, TGFbeta1, PAI-1 and also of TGF-beta1 related genes in 53 RCC and 30 normal kidney tissues by quantitative real-time RT-PCR (QRT-PCR). We further determined the BIGH3 protein levels in 52 cc-RCC paraffin-embedded tissue samples by immunohistochemistry. BIGH3 mRNA was found to be highly overexpressed in cc-RCC compared with normal tissues with an average ratio of 27. The mRNA levels of TGF-beta1 and PAI-1 were also detected at significantly elevated levels in these cancers. Immunohistochemical analysis of BIGH3 also revealed strong staining in the majority of the cc-RCC samples. In addition, the up-regulation of BIGH3 and PAI-1 was found to correlate with the clinicopathological parameters associated with a poorer patient outcome, whereas TGF-beta1 expression was determined to be unrelated to cancer progression. Strong BIGH3 staining thus tended to be associated with a poor prognosis. BIGH3 was also induced in some RCC cell lines by TGF-beta1 stimulation and this correlated well with PAI-1 up-regulation, suggesting that these enhancements are regulated by a similar mechanism in these tumors.

SOCS1 is an inducible host factor during HIV-1 infection and regulates the intracellular trafficking and stability of HIV-1 Gag.

Human immunodeficiency virus type 1 (HIV-1) utilizes the macromolecular machinery of the infected host cell to produce progeny virus. The discovery of cellular factors that participate in HIV-1 replication pathways has provided further insight into the molecular basis of virus-host cell interactions. Here, we report that the suppressor of cytokine signaling 1 (SOCS1) is an inducible host factor during HIV-1 infection and regulates the late stages of the HIV-1 replication pathway. SOCS1 can directly bind to the matrix and nucleocapsid regions of the HIV-1 p55 Gag polyprotein and enhance its stability and trafficking, resulting in the efficient production of HIV-1 particles via an IFN signaling-independent mechanism. The depletion of SOCS1 by siRNA reduces both the targeted trafficking and assembly of HIV-1 Gag, resulting in its accumulation as perinuclear solid aggregates that are eventually subjected to lysosomal degradation. These results together indicate that SOCS1 is a crucial host factor that regulates the intracellular dynamism of HIV-1 Gag and could therefore be a potential new therapeutic target for AIDS and its related disorders.

Functional roles of Fli-1, a member of the Ets family of transcription factors, in human breast malignancy.

The Ets family of transcription factors is implicated in malignant transformation and tumor progression, including invasion, metastasis and neo-angiogenesis. In the present study, we found that the Fli-1 gene, a member of the Ets family, was highly expressed in several breast cancer cell lines (MDA-MB231, MDA-MB436, BT-549 and HCC1395). To investigate the functional roles of Fli-1 in breast cancer malignancy, we introduced an expression plasmid containing full-length Fli-1 cDNA into MCF7 breast cancer cells in which endogenous expression of Fli-1 was barely detectable.Overexpression of Fli-1 in MCF7 cells led to inhibition of apoptosis induced by serum depletion or ultraviolet irradiation, although it did not affect cell growth rate in liquid media, colony formation in soft agar or the in vitro invasion capacity of the cells. Expression of Fli-1 and antiapoptotic bcl-2 was coordinately upregulated by serum depletion in MCF7 cells, and the upregulation was inhibited by treatment of the cells with a c-Jun-NH(2)-terminal kinase-specific inhibitor. Furthermore, expression of the bcl-2 gene and protein was enhanced in Fli-1-overexpressing MCF7 cells compared with mock-transfected cells. In addition, human bcl-2 promoter activity was transactivated by Fli-1. These results suggest that Fli-1 contributes to the malignancy of human breast cancer by inhibiting apoptosis through upregulated expression of the bcl-2 gene.

Activation of a system A amino acid transporter, ATA1/SLC38A1, in human hepatocellular carcinoma and preneoplastic liver tissues.

The expression of amino acid transporter (AT) mRNAs including A system (ATA1/SNAT1/SLC38A1, ATA2/SNAT2/SLC38A2 and ATA3/SNAT3/SLC38A4), L system (LAT1/SLC7A5 and LAT2/SLC7A8), and y+ (CAT2/SLC7A2) genes, were compared among hepatocellular carcinoma (HCC) and non-cancerous liver cells. Among them the ATA1 mRNA expression was significantly elevated in all HCC cell lines (HepG2, HLF, HuH7 and JHH4) examined compared with normal liver tissue. We further discovered that the expression of ATA1 mRNA was significantly activated in HCC tissues and also elevated in pre-malignant cirrhotic livers from HCC patients, compared with normal livers from non-HCC patients. The ATA1 protein was extensively accumulated in the cytoplasm of pre-malignant liver and most HCCs, while being weak or undetectably low in normal liver tissues. SiRNA-mediated suppression of endogenous ATA1 lowered the viability of HepG2 cells. Thus, the activation of ATA1 confers growth and survival advantages in pre-malignant and malignant liver lesions.

Gene expression signatures that can discriminate oral leukoplakia subtypes and squamous cell carcinoma.

The purpose of this study is to generate a classifier for oral squamous cell carcinoma (OSCC) and leukoplakias (LPs), and evaluate its diagnostic potential. In order to identify marker gene candidates, differential gene expression between LPs and OSCCs were examined by cDNA microarray. The expression of 118 marker gene candidates was further evaluated by quantitative reverse transcription-PCR (QRT-PCR) analyses of 27 OSCC and 19 LP tissues. We identified 12 up-regulated and 15 down-regulated marker genes in OSCCs compared to LPs. Using Fisher's linear discriminant analysis (LDA), we demonstrated that 11-gene predictors among this novel marker set could best distinguish OSCCs from LPs (>97% accuracy), whereas a further seven of these gene predictors could be utilized to distinguish higher grade (higher than moderate) from lower grade (lower than mild) dysplasias (>95% accuracy). These predictor gene sets provide multigene classifiers for the diagnosis of pre-cancerous to cancerous transition of oral malignancy.

Transformation-associated gene regulation by ATF6alpha during hepatocarcinogenesis.

We have previously reported that the endoplasmic reticulum (ER) stress-regulated transmembrane transcription factor 6 alpha (ATF6alpha) is implicated in the pathogenesis of hepatocellular carcinomas (HCCs). In order to further identify genes that are regulated by ATF6alpha, the global gene expression profiles of the ATF6alpha-transfected and untransfected HCC cell line, HLF, were analyzed. These results were then compared with the differential gene expression patterns of poorly differentiated HCC and control non-tumorous liver tissue. Our findings demonstrate that at least 18 genes are specifically upregulated by ATF6alpha, while another UPR mediator, XBP1 or ER-stress inducer, thapsigargin could partially stimulate or even repress some of them in HCC cells. Moreover, six of these identified genes contain potential ER stress-responsive elements and/or unfolded protein response elements in their 5' regulatory regions.

Tricyclic pharmacophore-based molecules as novel integrin alpha(v)beta3 antagonists. Part 1: design and synthesis of a lead compound exhibiting alpha(v)beta3/alpha(IIb)beta3 dual antagonistic activity.

In order to generate novel compounds with integrin alpha(v)beta3-antagonistic activity together with antiplatelet activity, tricyclic pharmacophore-based molecules were designed and synthesized. Although piperazine-containing compounds initially prepared were selective alpha(IIb)beta3 antagonists, replacement of piperazine with piperidine furnished a potent alpha(v)beta3/alpha(IIb)beta3 dual antagonist. Structure-activity relationship (SAR) studies provided clues for further development of tricyclic pharmacophore-based integrin antagonists.

Tricyclic pharmacophore-based molecules as novel integrin alpha(v)beta3 antagonists. Part 2: synthesis of potent alpha(v)beta3/alpha(IIb)beta3 dual antagonists.

We synthesized 4-aminopiperidine derivatives of our prototype integrin alpha(v)beta3 antagonist 1 in an attempt to increase the activity and water solubility. Introduction of one or two hydrophilic moieties into the central aromatic ring and/or the benzene ring at the C-terminus of 1 increased water solubility and enhanced inhibition of cell adhesion. The results of a structure-activity relationships (SAR) study indicated that the torsion angle between the central aromatic ring and the piperidine ring, and the acidity at the sulfonamide moiety, might be important for alpha(v)beta3 receptor binding activity. Some of these compounds are novel and potent alpha(v)beta3/alpha(IIb)beta3 dual antagonists with acceptable water solubility and a satisfactory early absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile.

Complex formations involving both SP-1 and SP-3 at the transcriptional regulatory sequence correlate with the activation of the Keratin 14 gene in human oral squamous cell carcinoma cells.

We have previously reported that significantly higher levels of Keratin 14 (Ker-14) was observed in oral squamous cell carcinoma (OSCC) and severely dysplastic tissues, whereas this expression was reversed in hyperplasia and in mild to moderate dysplasia. In this study, the mechanism of Keratin 14 activation in oral squamous cell carcinoma (OSCC) cell lines (HSC-2, HSC-3 and Ca9-22) was investigated. Reporter analysis demonstrated that an upstream region (-1759/-1629) accounted for efficient promoter activity. Furthermore, electromobility sift and supershift assay demonstrated that interactions of the SP-1/SP-3 complex at the elements resided in -1737/-1702 and -1680/-1652 and may be essential for this activation in OSCC cells.

Differential expression of the keratin-4, -13, -14, -17 and transglutaminase 3 genes during the development of oral squamous cell carcinoma from leukoplakia.

To identify differentially expressed genes during the development of oral malignancy, differential display, northern blotting, reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analyses were undertaken using oral squamous cell carcinoma (OSCC) and leukoplakia tissues. Significantly higher levels of keratin (Ker)-14 and -17 mRNAs, combined with lower levels of Ker-4, Ker-13 and transglutaminase 3 (TG-3) transcripts, were observed in OSCC and severely dysplastic tissues, whereas this expression profile was reversed in hyperplasia and in mild to moderate dysplasia. The expression of Ker-4 and Ker-13 was elevated in density-arrested OSCC cell lines (Ca9-22, HSC-2, -3 and -4) but the expression of Ker-17 mRNA was elevated in these cells, regardless of the growth conditions. In addition, Ker-4 and Ker-13 proteins were predominantly expressed in moderate dysplasia and hyperplasia, whereas Ker-17 was markedly expressed in OSCC tissues. The expression patterns of these genes could therefore be an important determinant of the manifestation of oral malignancy.

Purification and characterization of Acremonium implicatum alpha-glucosidase having regioselectivity for alpha-1,3-glucosidic linkage.

alpha-Glucosidase with a high regioselectivity for alpha-1,3-glucosidic linkages for hydrolysis and transglucosylation was purified from culture broth of Acremonium implicatum. The enzyme was a tetrameric protein (M.W. 440,000), of which the monomer (M.W. 103,000; monomeric structure was expected from cDNA sequence) was composed of two polypeptides (M.W. 51,000 and 60,000) formed possibly by posttranslational proteolysis. Nigerose and maltose were hydrolyzed by the enzyme rapidly, but slowly for kojibiose. The k(0)/K(m) value for nigerose was 2.5-fold higher than that of maltose. Isomaltose was cleaved slightly, and sucrose was not. Maltotriose, maltotetraose, p-nitrophenyl alpha-maltoside and soluble starch were good substrates. The enzyme showed high affinity for maltooligosaccharides and p-nitrophenyl alpha-maltoside. The enzyme had the alpha-1,3- and alpha-1,4-glucosyl transfer activities to synthesize oligosaccharides, but no ability to form alpha-1,2- and alpha-1,6-glucosidic linkages. Ability for the formation of alpha-1,3-glucosidic linkage was two to three times higher than that for alpha-1,4-glucosidic linkage. Eight kinds of transglucosylation products were synthesized from maltose, in which 3(2)-O-alpha-nigerosyl-maltose and 3(2)-O-alpha-maltosyl-maltose were novel saccharides.

Mouse testis transcriptome revealed using serial analysis of gene expression.

We applied serial analysis of gene expression (SAGE) to the mouse testis to reveal the global gene expression profile and to identify senescence-dependent changes in that profile. A total of 61,929 SAGE tags, including 19,323 unique tags, were obtained from 3- and 29-month-old BDF1 mice and 14-month-old SAMP1 mice. Genes highly expressed in the testis included those associated with spermatogenesis, protein metabolism, energy metabolism, growth and differentiation, and signal transduction. Testes from old mice of both strains appeared atrophied. Morphological examination of aged testes revealed extremely thin seminiferous epithelia and significantly decreased numbers of spermatids and spermatocytes. Despite the physical deterioration, no gross changes in the gene expression profile were apparent in the testes of old BDF1 mice. However, in 14-month-old SAMP1 mice, protamine 2 gene transcription was approximately 50% lower than in BDF1 mice. This reduction may be associated with the oligozoospermia and early decline in reproductive performance of SAMP1 mice. Our SAGE results are the first quantitative gene expression profile of the mouse testis and provide a reliable transcriptome reference for this organ.