Discovery and characterization of NK13650s, naturally occurring p300-selective histone acetyltransferase inhibitors.

The histone acetyltransferase (HAT) activity of p300 is essential for androgen receptor (AR) function. Androgen-independent prostate cancer cells require AR-mediated transcriptional activation for their growth. These observations indicate that p300 HAT is a promising target to overcome such hormone-resistant cancer cells. We sought p300 HAT inhibitors among microbial metabolites. By culturing a production strain belonging to Penicillium, we identified two new compounds, NK13650A and NK13650B, which were obtained as specific p300 HAT inhibitors. Structural analyses of these compounds elucidated that NK13650s have novel chemical structures comprising several amino acids and citrate. We applied a newly developed biosynthesis-based method to reveal the absolute configuration at the citrate quaternary carbon. This was accomplished by feeding a (13)C-labeled biosynthetic precursor of citrate. NK13650s selectively inhibited the activity of p300 HAT but not that of Tip60 HAT. NK13650s showed inhibitory activity against agonist-induced AR transcriptional activation, and NK13650A treatment inhibited hormone-dependent and -independent growth of prostate cancer cells.

Involvement of non­triple helical type VI collagen α1 chain, NTH α1(VI), in the proliferation of cancer cells.

It has been reported that a polypeptide encoded by collagen type VI alpha 1 chain (COL6A1), one of the three α chains of type VI collagen, is strongly associated with the migration and invasion of highly metastatic human pancreatic cancer BxPC­M8 cells and excessive proliferation of LNCaP cells. We previously reported that non­triple helical type VI collagen α1 chain, NTH α1(VI), a non­triple helical polypeptide encoded by COL6A1, is not derived from type VI collagen and exists in cancer cell­conditioned media. Therefore, NTH α1(VI) may be involved in cancer cell migration, invasion, and proliferation. The active entity that promotes cellular behaviors in cancer remains unclear. Thus, we predicted that NTH α1(VI) has cancer­promoting activity, such as the ability to induce cell proliferation. This study was conducted to examine whether NTH α1(VI) and/or its derived peptides are involved in cancer cell proliferation. Highly metastatic human pancreatic S2­VP10 cells were used to explore the potential of COL6A1 knockdown in reducing cell proliferation. Moreover, S2­VP10 conditioned medium was assessed after molecular size­fractionation to determine whether the inhibitory effect of COL6A1 knockdown could be rescued by the medium. We showed that S2­VP10­conditioned medium contained COL6A1 polypeptide, but not COL6A2, suggesting that COL6A1 in the conditioned medium of S2­VP10 cells reflects the presence of NTH α1(VI). COL6A1 knockdown repressed S2­VP10 cell proliferation and this repression was rescued using the conditioned medium of S2­VP10 cells. The fraction of conditioned medium containing peptides smaller than 10 kDa rescued the inhibitory effect; however, the fraction containing polypeptides larger than 10 kDa, including NTH α1(VI), did not show rescue activity, indicating that NTH α1(VI) fragmentation is necessary for enhanced cancer cell proliferation. In conclusion, fragmentation of NTH α1(VI) into peptides <10 kDa is required for its cancer cell proliferation­promoting activity.

Suppression of mutant androgen receptors by flutamide.

OBJECTIVES: To examine the effects of flutamide and hydroxyflutamide on the transactivation of mutant androgen receptors. METHODS: Androgen-independent human prostate cancer cell line PC3 was transfected with plasmids expressing wild-type, W741C mutant, T877A mutant or W741C+T877A mutant androgen receptors. The effects of bicalutamide, hydroxyflutamide or flutamide on the basal and dihydrotestosterone-induced transcriptional activities of the wild-type and mutant androgen receptors were evaluated by luciferase assays using a reporter plasmid containing the prostate-specific antigen (PSA) promoter. The effects of the antiandrogens on the transcription and translation of the PSA gene in LNCaP cells expressing a mutant (T877A) androgen receptor were assessed by real-time reverse transcription-polymerase chain reaction and radioimmunoassays. Affinity of the antiandrogens to each androgen receptor construct was evaluated by ligand-binding assay. RESULTS: Flutamide, but not hydroxyflutamide, successfully suppressed the transcription of all of the mutant androgen receptors examined in this study and also showed suppressive effects on PSA secretion by LNCaP cells treated with dihydrotestosterone. These inhibitory effects were probably not the result of competitive inhibition by flutamide given its low affinity to the androgen receptor constructs. CONCLUSIONS: Flutamide, with its suppressive effects on mutant androgen receptors, may be an alternative to conventional antiandrogens for hormone refractory prostate cancer.

Identification of a common epitope in the sequences of COL4A1 and COL6A1 recognized by monoclonal antibody #141.

Identification of a type IV collagen α1 polypeptide in non-triple helical form [NTH α1(IV)], possibly involved in angiogenesis, introduces the further possibility of the existence of non-triple helical forms of other collagen chains. We previously reported that an anti-NTH α1(IV) monoclonal antibody #141 recognizes not only NTH α1(IV) but also a novel non-triple helical collagen polypeptide NTH α1(VI) encoded by COL6A1. In this study, we identified the recognition sequence in order to better understand the properties of antibody #141 and provide clues regarding the biological function of the two non-triple helical molecules. Additionally, we determined the common epitope between COL4A1 and COL6A1 as PXXGXPGLRG, with surface plasmon resonance analyses revealing KD values for the COL4A1 epitope as 5.56±1.81×10-9 M and for the COL6A1 epitope as 7.15±0.44×10-10 M. The specific recognition of NTH α1(IV) and NTH α1(VI) by antibody #141 can be explained by the common epitope sequence. Moreover, epitope localization supports previous finding that NTH α1(IV) and NTH α1(VI) differ in conformation from the α1 chains in triple-helical type IV and type VI collagen. These findings suggest that antibody #141 might be useful for diagnosis of type VI collagen myopathies.

Type VI collagen α1 chain polypeptide in non-triple helical form is an alternative gene product of COL6A1.

Expression of type IV collagen α1 chain in non-triple helical form, NTH α1(IV), is observed in cultured human cells, human placenta and rabbit tissues. Biological functions of NTH α1(IV) are most likely to be distinct from type IV collagen, since their biochemical characteristics are quite different. To explore the biological functions of NTH α1(IV), we prepared some anti-NTH α1(IV) antibodies. In the course of characterization of these antibodies, one antibody, #141, bound to a polypeptide of 140 kDa in size in addition to NTH α1(IV). In this study, we show evidence that the 140 kDa polypeptide is a novel non-triple helical polypeptide of type VI collagen α1 chain encoded by COL6A1, or NTH α1(VI). Expression of NTH α1(VI) is observed in supernatants of several human cancer cell lines, suggesting that the NTH α1(VI) might be involved in tumourigenesis. Reactivity with lectins indicates that sugar chains of NTH α1(VI) are different from those of the α1(VI) chain in triple helical form of type VI collagen, suggesting a synthetic mechanism and a mode of action of NTH α1(VI) is different from type VI collagen.

Preparation and partial characterization of monoclonal antibodies specific for the nascent non-triple helical form of the type IV collagen alpha 1 chain.

This report describes the preparation and partial characterization of monoclonal antibodies that are reactive specifically with the nascently produced non-triple helical form of the type IV collagen α1 chain, designated as NTH α1(IV). These antibodies were nonreactive with the α1 chain of the type IV collagen in the triple-helical conformation. Three antibodies, #141, #179 and #370, with different epitopes in NTH α1(IV) were found to be reactive with the nascent polypeptide secreted from human normal cells and a human carcinoma cell line. The antibodies with different epitopes may provide a key method for elucidating the physiological function and tissue distribution of NTH α1(IV), which is distinct from the chain derived from triple-helical type IV collagen.

Metabolism and hepatic toxicity of flutamide in cytochrome P450 1A2 knockout SV129 mice.

BACKGROUND: Flutamide, a nonsteroidal antiandrogen used for treatment of prostate cancer, causes a temporary increase in transaminase and in some cases severe liver dysfunction. It is dominantly metabolized by cytochrome P450 (CYP) 1A2 into 2-hydroxyflutamide (OH-flutamide), which has stronger antiandrogenic activity without obvious cytotoxicity to cultured hepatocytes. We hypothesized that another subsidiary metabolite might be responsible for induction of hepatotoxicity. METHODS: Flutamide was administered daily to CYP1A2 knockout mice and parental SV129 mice to compare pharmacokinetics and appearance of hepatic toxicity. RESULTS: In the CYP1A2 knockout mice, the plasma concentration of flutamide maintained at a high level and OH-flutamide stayed low; a higher amount of FLU-1, an alternative metabolite of flutamide, was detected in urine. Simple repetitive administration of 800 mg/kg of flutamide for 28 days to CYP1A2 knockout mice did not show abnormal elevation of plasma alanine aminotransferase (ALT). However, after the knockout mice were fed with an amino acid-deficient diet for 2 weeks, which reduced the glutathione (GSH) content to 27% of the initial, administration of 400 mg/kg of flutamide increased ALT to over 200 IU/l and histopathologically moderate hepatitis developed. Since FLU-1 itself did not show cytotoxicity or reduce GSH content in vitro, a further metabolized molecule must cause the hepatotoxicity. CONCLUSIONS: Blockade of CYP1A2 produced an unknown potential hepatotoxic molecule through FLU-1, and GSH might play an important role in diminishing the reactive hepatotoxic metabolite.

The presence of both the amino- and carboxyl-terminal domains in the AR is essential for the completion of a transcriptionally active form with coactivators and intranuclear compartmentalization common to the steroid hormone receptors: a three-dimensional imaging study.

To clarify the physiological significance of the intranuclear speckled distribution, or foci formation, of liganded steroid receptors, the subnuclear distribution of green (GFP), yellow (YFP), and cyan (CFP) fluorescent protein-tagged receptors and coactivators was investigated. The foci formation of 5 alpha-dihydrotestosterone (DHT)-bound AR-GFP in COS7 cells was abolished by the cotransfection of a CBP Delta (118-2393) fragment eliciting a dominant negative effect on the transactivation capacity of the AR. The N-terminal AR fragment (AR-AF-1-YFP), which has a strong constitutive transactivation function, formed foci without DHT, whereas the C-terminal AR fragment (AR-AF-2-CFP), which has a quite low transactivation function, was distributed homogeneously even in the presence of DHT. The reporter gene assay showed a synergism between the transactivation functions of AR-AF-1 and AR-AF-2. This synergism was not reflected by the above two-dimensional imaging. In contrast, a three-dimensional imaging method clearly showed a difference in the intranuclear spatial distribution. The DHT-bound wild-type AR-GFP alone or AR-AF-1-YFP plus DHT-bound AR-AF-2-CFP was distributed as approximately 300 discrete spots in one nucleus, whereas AR-AF-1-YFP alone was distributed as one volume in a reticular pattern. Furthermore, not only AR but also the glucocorticoid receptor-YFP, ER alpha -GFP, and YFP-tagged SRC-1, TIF2, and CBP were found to be accumulated in identical spots in the presence of ligand. All of the above results indicate that CBP is one of the factors essential for foci formation of the AR, and may propose the hypothesis that transcriptionally activated steroid receptors, regardless of the type of receptor, are transferred to common compartments (foci) and form a complex with coactivators, and this process is essential to full transactivation.

A novel compound, NK150460, exhibits selective antitumor activity against breast cancer cell lines through activation of aryl hydrocarbon receptor.

Antiestrogen agents are commonly used to treat patients with estrogen receptor (ER)-positive breast cancer. Tamoxifen has been the mainstay of endocrine treatment for patients with early and advanced breast cancer for many years. Following tamoxifen treatment failure, however, there are still limited options for subsequent hormonal therapy. We discovered a novel compound, NK150460, that inhibits 17ß-estradiol (E2)-dependent transcription without affecting binding of E2 to ER. Against our expectations, NK150460 inhibited growth of not only most ER-positive, but also some ER-negative breast cancer cell lines, while never inhibiting growth of non-breast cancer cell lines. Cell-based screening using a random shRNA library, identified aryl hydrocarbon receptor nuclear translocator (ARNT) as a key gene involved in NK150460's antitumor mechanism. siRNAs against not only ARNT but also its counterpart aryl hydrocarbon receptor (AhR) and their target protein, CYP1A1, dramatically abrogated NK150460's growth-inhibitory activity. This suggests that the molecular cascade of AhR/ARNT plays an essential role in NK150460's antitumor mechanism. Expression of ERα was decreased by NK150460 treatment, and this was inhibited by an AhR antagonist. Unlike two other AhR agonists now undergoing clinical developmental stage, NK150460 did not induce histone H2AX phosphorylation or p53 expression, suggesting that it did not induce a DNA damage response in treated cells. Cell lines expressing epithelial markers were more sensitive to NK150460 than mesenchymal marker-expressing cells. These data indicate that NK150460 is a novel AhR agonist with selective antitumor activity against breast cancer cell lines, and its features differ from those of the other two AhR agonists.

Activation function-1 domain of androgen receptor contributes to the interaction between two distinct subnuclear compartments.

The nucleus contains different sets of functional compartments often called "speckles". The splicing factor compartment (SFC) has been speculated to consist of SFs and transcription factors, which thus make transcription-splicing coupling possible at the periphery of SFC. Androgen receptor (AR), as well as glucocorticoid receptor (GR), is unique since most, if not all, of its activities are mediated via the constitutive activity of the activation function-1 (AF-1) function. Transcriptionally active AR produces 250-400 subnuclear fine speckles11 shared with GR or estrogen receptor (ER), which colocalize with chiefly activation function-2 (AF-2)-interacting p160 family- or CBP-related speckles. We herein report the isolation of ANT-1 (AR N-terminal domain (NTD) transactivating protein-1) enhancing autonomous AF-1 transactivation function of AR or GR, but not of estrogen receptor alpha (ERalpha). The ANT-1 was identical to a binding protein of human splicing factor U5 snRNP (U5 snRNP-associated protein). ANT-1 was compartmentalized into 15-20 coarse SFC speckles which were spatially distinct from but surrounded by the AR compartments. Our results suggest that ANT-1 may play a key role in the molecular interaction between two spatially distinct subnuclear compartments in a receptor-specific fashion, and thereby induce the strong autonomous transactivation functions either of AR- or GR-AF-1.