Amidation inhibitors 4-phenyl-3-butenoic acid and 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester are novel HDAC inhibitors with anti-tumorigenic properties.

4-Phenyl-3-butenoic acid (PBA) is an inhibitor of peptidylglycine alpha-amidating monooxygenase with anti-inflammatory properties that has been shown to inhibit the growth of ras-mutated epithelial and human lung carcinoma cells. In this report, we show that PBA also increases the acetylation levels of selected histone subtypes in a dose and time dependent manner, an effect that is attributable to the inhibition of histone deacetylase (HDAC) enzymes. Comparison studies with the known HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) using high resolution two-dimensional polyacrylamide gels and Western analysis provide evidence that PBA acts as an HDAC inhibitor within cells. PBA and a more potent amidation inhibitor, 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me), inhibit HDAC enzymes in vitro at micromolar concentrations, with IC50 values approximately 30 fold lower for AOPHA-Me than PBA for selected HDAC isoforms. Overall, these results indicate that PBA and AOPHA-Me are novel anti-tumorigenic HDAC inhibitors.

Comparative effects of 4-phenyl-3-butenoic acid and vorinostat on cell growth and signaling.

BACKGROUND/AIM: 4-phenyl-3-butenoic acid (PBA) is a small-molecule anti-inflammatory agent, which has been shown to inhibit growth, increase gap junction intercellular communication and modulate activation of p38 mitogen-activated protein kinase (p38 MAPK) and c-jun n-terminal kinase (JNK) in tumorigenic cells at concentrations that do not similarly affect non-tumorigenic cells. Vorinostat is an anticancer agent structurally similar to PBA. The purpose of this study was to compare the effects of these two agents on JNK and p38 activation, cell growth and gap junction intercellular communication (GJIC). MATERIALS AND METHODS: Cell growth, GJIC and western blot analyses were performed utilizing tumorigenic WBras1 and H2009 human carcinoma cells, and non-tumorigenic WBneo3 and human bronchial epithelial (HBE) cells. RESULTS: Both compounds significantly inhibited WBras1 and H2009 tumorigenic cell growth and increased GJIC in WBras1 cells, as previously reported for PBA. Under similar conditions, both compounds increased phosphorylation of p38 MAPK in tumorigenic but not in non-tumorigenic cells and decreased phosphorylation of JNK in tumorigenic cells. However, a decrease in phosphorylation of JNK occurred in non-tumorigenic WBras1 cells following vorinostat treatment but not PBA treatment. Both compounds showed a selective growth inhibition of H2009 human carcinoma over normal HBE lung cells but, unlike PBA, vorinostat significantly decreased cell growth in WBneo3 cells. CONCLUSION: Overall, PBA exhibited similar effects to vorinostat in tumorigenic cells, while also showing reduced effects on JNK phosphorylation and growth in non-tumorigenic cells compared to ras-transformed cells.

Inhibition of JNK and p38 MAPK phosphorylation by 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester and 4-phenyl-butenoic acid decreases substance P-induced TNF-α upregulation in macrophages.

The interactions between the immune and nervous systems play an important role in immune and inflammatory conditions. Substance P (SP), the undecapeptide RPKPQQFFGLM-NH2, is known to upregulate the production of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α. We report here that 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid methyl ester (AOPHA-Me) and 4-phenyl-3-butenoic acid (PBA), two anti-inflammatory compounds developed in our laboratory, reduce SP-stimulated TNF-α expression in RAW 264.7 macrophages. We also show that AOPHA-Me and PBA both inhibit SP-stimulated phosphorylation of JNK and p38 MAPK. Furthermore, molecular modeling studies indicate that both AOPHA-Me and PBA dock at the ATP binding site of apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPKs upstream of both JNK and p38 MAPK, with predicted interaction energies of -7.0 kcal/mol and -5.9 kcal/mol, respectively; this binding overlaps with that of staurosporine, a known inhibitor of ASK1. Taken together, these findings suggest that AOPHA-Me and PBA inhibition of TNF-α expression in SP-stimulated RAW 264.7 macrophages is a consequence of the inhibition of JNK and p38 MAPK phosphorylation. We have previously shown that AOPHA-Me and PBA inhibit the amidative bioactivation of SP, which also would be expected to decrease formation of pro-inflammatory cytokines. It is conceivable that this dual action of inhibiting amidation and MAPK phosphorylation may be of some advantage in enhancing the anti-inflammatory activity of a therapeutic molecule.

A Cell-Cell Communication Marker for Identifying Targeted Tumor Therapies.

Cell-cell communication through gap junctions is aberrant or absent in a majority of human cancer cells, compared to cells in corresponding normal tissues. This and other evidence has led to the hypothesis that gap junction channels, comprised of connexin proteins, are important in growth control and cancer progression. The major goal of this ongoing study was to identify bioactive compounds that specifically upregulate gap junction channel-mediated cell-cell communication as potential anti-tumor therapies. Control of cell-cell communication is linked to growth regulatory intracellular signaling pathways; we therefore further aimed to identify signaling pathways modulated by these compounds in order to assess their potential as targeted anti-tumor therapies. Compounds were screened for their ability to upregulate gap junction-mediated cell-cell communication by using a fluorescent dye transfer assay to measure cell-cell communication between tumor promoter-treated astroglial cells or ras-transformed epithelial cells. Western blotting using connexin-specific and phosphorylation site-specific antibodies was used to monitor phosphorylation changes in signaling pathway proteins. Our results identified three compounds that upregulate gap junction-mediated cell-cell communication in our screening assays, chaetoglobosin K(ChK), 4-phenyl-3-butenoic acid (PBA) and the methyl ester of PBA (PBA-Me). Further analyses demonstrated that in tumorigenic cells, ChK downregulates phosphorylation of Akt kinase, an enzyme in the PI3-kinase signaling pathway that is found to be upregulated in a number of human cancers, on a key activation site. However, ChK did not inhibit PI-3 kinase in vitro as did the classic PI-3 kinase inhibitor, Wortmannin. PBA and PBA-Me were found to upregulate phosphorylation of p38 MAPK on a key activation site in tumorigenic cells, which is downregulated in several human cancer cell types. ChK and PBA also decreased activation of SAPK/JNK, another kinase found to be upregulated in a number of human cancers. These studies highlight the potential of monitoring gap junction intercellular communication for identifying experimental anti-tumor compounds.

p38 MAPK activation, JNK inhibition, neoplastic growth inhibition, and increased gap junction communication in human lung carcinoma and Ras-transformed cells by 4-phenyl-3-butenoic acid.

Human lung neoplasms frequently express mutations that down-regulate expression of various tumor suppressor molecules, including mitogen-activated protein kinases such as p38 MAPK. Conversely, activation of p38 MAPK in tumor cells results in cancer cell cycle inhibition or apoptosis initiated by chemotherapeutic agents such as retinoids or cisplatin, and is therefore an attractive approach for experimental anti-tumor therapies. We now report that 4-phenyl-3-butenoic acid (PBA), an experimental compound that reverses the transformed phenotype at non-cytotoxic concentrations, activates p38 MAPK in tumorigenic cells at concentrations and treatment times that correlate with decreased cell growth and increased cell-cell communication. H2009 human lung carcinoma cells and ras-transformed rat liver epithelial cells treated with PBA showed increased activation of p38 MAPK and its downstream effectors which occurred after 4 h and lasted beyond 48 h. Untransformed plasmid control cells showed low activation of p38 MAPK compared to ras-transformed and H2009 carcinoma cells, which correlates with the reduced effect of PBA on untransformed cell growth. The p38 MAPK inhibitor, SB203580, negated PBA's activation of p38 MAPK downstream effectors. PBA also increased cell-cell communication and connexin 43 phosphorylation in ras-transformed cells, which were prevented by SB203580. In addition, PBA decreased activation of JNK, which is upregulated in many cancers. Taken together, these results suggest that PBA exerts its growth regulatory effect in tumorigenic cells by concomitant up-regulation of p38 MAPK activity, altered connexin 43 expression, and down-regulation of JNK activity. PBA may therefore be an effective therapeutic agent in human cancers that exhibit down-regulated p38 MAPK activity and/or activated JNK and altered cell-cell communication.