Reveromycin C

Earlier work in our laboratory demonstrated that naturally occurring reveromycin A (Rev A) causes apoptosis in osteoclasts without accompanying necrosis. Rev A death effects in both normal and diseased joint cells were investigated in this study. A dose of 10 μM Rev A did not cause apoptosis nor necrosis in monolayer chondrocytes, even at pH 6.8, a pH mimicking that of an inflamed joint. In contrast, at the acidic pH Rev A did induce significant apoptosis (fourfold increase at 48 h of treatment, P < 0.005) in normal synoviocytes without accompanying necrosis. Western blot of the normal synoviocyte proteins revealed that cytochrome c levels were not significantly changed over the time course of treatment nor did caspase 8 activity increase; therefore, Rev A appears to exert this apoptotic effect through a mechanism independent of the classical intrinsic and extrinsic pathways. Fibroblast-like synoviocytes isolated from rheumatoid arthritis patients (RAFLS) as well as normal human fibroblast-like synoviocytes (NHFLS), cells known to play key roles in arthritic joint pathology, were also subjected to Rev A treatment at both physiologic and acidic pH's. Neither apoptosis nor necrosis was induced in either RAFLS or NHFLS. Parallel mitomycin C treatment of NHFLS induced both apoptosis and necrosis. Comparative structure-activity analyses of Rev A and mitomycin C revealed that Rev A is less likely to cross the cell membrane at near neutral pH. Collectively the data reveal that a physiological dose of Rev A under acidic conditions induces normal synoviocytes to undergo apoptosis while pathologic fibroblast-like synoviocytes are resistant to apoptosis and necrosis.

Attention has recently focused on the critical role of inflammatory responses in the tumor stroma that provide favorable conditions for cancer-cell growth and invasion/metastasis. In particular, macrophages recruited into the tumor stroma and activated, known as tumor-associated macrophages, are suggested to promote tumorigenesis. In this study, we examined the effect of a decrease in the number of monocytes/macrophages in peripheral blood and the tumor stroma on the development of bone and muscle metastases by lung cancer cells. Treatment with clodronate encapsulated by liposomes (Cl(2)MDP-LIP) has been developed for the depletion of monocytes/macrophages in an animal model. Subcutaneous administration of Cl(2)MDP-LIP markedly reduced the number of monocytes in peripheral blood, resulting in efficient suppression of both bone metastasis and muscle metastasis when lung cancer HARA-B cells were injected into the left cardiac ventricle of mice. Treatment with Cl(2)MDP-LIP significantly reduced the number of macrophages in tumors and the number of osteoclasts in bone marrow, as well as peripheral monocytes in mice harboring lung cancer cells. In contrast, treatment with an osteoclast-targeting antibiotic, reveromycin A, inhibited bone metastasis by lung cancer cells, but not muscle metastasis. The survival of human macrophages in culture was found to be specifically blocked by Cl(2)MDP-LIP, but not by reveromycin A. Cl(2)MDP-LIP thus exerted antimetastatic effects in both bone and muscle whereas reveromycin A did so only in bone. Liposome-encapsulated bisphosphonate may modulate metastasis through decreasing the number of monocytes/macrophages in both peripheral blood and the tumor stroma, suggesting that tumor-associated macrophages might be suitable targets for antimetastatic therapy.

Understanding the biosynthetic mechanism of the atypical polyketide extender unit is important for the development of bioactive natural products. Reveromycin (RM) derivatives produced by Streptomyces sp. SN-593 possess several aliphatic extender units. Here, we studied the molecular basis of 2-alkylmalonyl-CoA formation by analyzing the revR and revS genes, which form a transcriptional unit with the revT gene, a crotonyl-CoA carboxylase/reductase homolog. We mainly focused on the uncharacterized adenylate-forming enzyme (RevS). revS gene disruption resulted in the reduction of all RM derivatives, whereas reintroduction of the gene restored the yield of RMs. Although RevS was classified in the fatty acyl-AMP ligase clade based on phylogenetic analysis, biochemical characterization revealed that the enzyme catalyzed the middle chain fatty acyl-CoA ligase (FACL) but not the fatty acyl-AMP ligase activity, suggesting the molecular evolution for acyl-CoA biosynthesis. Moreover, we examined the in vitro conversion of fatty acid into 2-alkylmalonyl-CoA using purified RevS and RevT. The coupling reaction showed efficient conversion of hexenoic acid into butylmalonyl-CoA. RevS efficiently catalyzed C8-C10 middle chain FACL activity; therefore, we speculated that the acyl-CoA precursor was truncated via β-oxidation and converted into (E)-2-enoyl-CoA, a RevT substrate. To determine whether the β-oxidation process is involved between the RevS and RevT reaction, we performed the feeding experiment using [1,2,3,4-(13)C]octanoic acid. (13)C NMR analysis clearly demonstrated incorporation of the [3,4-(13)C]octanoic acid moiety into the structure of RM-A. Our results provide insight into the role of uncharacterized RevS homologs that may catalyze middle chain FACL to produce a unique polyketide extender unit.