Farnesyltransferase Inhibitor Manumycin Research Articles

Identification of proteasome inhibitors using analysis of gene expression profiles

Inhibitors of the 20S proteasome such as bortezomib (Velcade®) and carfilzomib (Kypriolis®) are in clinical use for the treatment of patients with multiple myeloma and mantle cell lymphoma. In an attempt to identify novel inhibitors of the ubiquitin-proteasome system (UPS) we used the connectivity map (CMap) resource, based on alterations of gene expression profiles by perturbagens, and performed COMPARE analyses of drug sensitivity patterns in the NCI60 panel. Cmap analysis identified a large number of small molecules with strong connectivity to proteasome inhibition, including both well characterized inhibitors of the 20S proteasome and molecules previously not described to inhibit the UPS. A number of these compounds have been reported to be cytotoxic to tumor cells and were tested for their ability to decrease processing of proteasome substrates. The antibiotic thiostrepton and the natural products celastrol and curcumin induced strong accumulation of polyubiquitinated proteasome substrates in exposed cells. Other compounds elicited modest increases of proteasome substrates, including the protein phosphatase inhibitor BCI–Cl and the farnesyltransferase inhibitor manumycin A, suggesting that these compounds inhibit proteasome function. Induction of chaperone expression in the absence of proteasome inhibition was observed by a number of compounds, suggesting other effects on the UPS. We conclude that the combination of bioinformatic analyses and cellular assays resulted in the identification of compounds with potential to inhibit the UPS.

Ram1 gene, encoding a subunit of farnesyltransferase, contributes to growth, antifungal susceptibility to amphotericin B of Aspergillus fumigatus.

Farnesylation, which is catalyzed by farnesyltransferase, is an important posttranslational process. The function of farnesyltransferase has been previously explored in Cryptococcus neoformans and Candida albicans. Aspergillus fumigatus is an important human opportunistic fungal pathogen in immunocompromised patients. Here we discover the role of the ram1 gene, encoding the β-subunit of farnesyltransferase in A. fumigatus, in the fungal growth and antifungal susceptibility. In this study the ram1 gene was disrupted using A. tumefaciens-mediated transformation. The morphology and radial growth of Δram1 were observed. Assays of disk diffusion and broth microdilution were used to determine the susceptibility of Δram1 mutant to commonly clinical used antifungals and the farnesyltransferase inhibitor manumycin A. Deletion of ram1 resulted in a reduced radial growth of A. fumigatus but did not affect the microscopic morphology. Δram1 showed increased susceptibility to the antifungal amphotericin B; however, its susceptibility to azoles and caspofungin was the same to that to the parental strain. Our data indicate that farnesyltransferase is a potential target for design new antifungal agents.

Stimulating Effect of Manumycin A on Suicidal Erythrocyte Death

Background/Aims: The streptomycete derived farnesyltransferase inhibitor Manumycin A triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. The present study explored whether Manumycin A could similarly stimulate eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include Ca²⁺ entry as well as activation of staurosporine sensitive protein kinase C and SB203580 sensitive p38 kinase. The present study explored, whether Manumycin A induces eryptosis and, if so, to shed some light on the mechanisms involved. Methods: Phosphatidylserine abundance at the human erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, and hemolysis from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Manumycin A (≥ 5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly incrased hemolysis. The effect of Manumycin A on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺, by addition of staurosporine (1 µM) and by addition of SB203580 (2 µM). Conclusions: Manumycin A triggers hemolysis, cell shrinkage and phospholipid scrambling of the human erythrocyte cell membrane. The effect on cell membrane scrambling was in part but not fully dependent on entry of extracellular Ca²⁺, as well as activity of staurosporine and SB203580 sensitive kinases.

Enhanced Antiproliferative and Apoptotic Response of HT-29 Adenocarcinoma Cells to Combination of Photoactivated Hypericin and Farnesyltransferase Inhibitor Manumycin A

Several photodynamically-active substances and farnesyltransferase inhibitors are currently being investigated as promising anticancer drugs. In this study, the combined effect of hypericin (the photodynamically-active pigment from Hypericum perforatum) and selective farnesyltransferase inhibitor manumycin (manumycin A; the selective farnesyltransferase inhibitor from Streptomyces parvulus) on HT-29 adenocarcinoma cells was examined. We found that the combination treatment of cells with photoactivated hypericin and manumycin resulted in enhanced antiproliferative and apoptotic response compared to the effect of single treatments. This was associated with increased suppression of clonogenic growth, S phase cell cycle arrest, elevated caspase-3/7 activity and time-dependent total cleavage of procaspase-3 and lamin B, cleavage of p21Bax into p18Bax and massive PARP cleavage. Moreover, we found that the apoptosis-inducing factor is implicated in signaling events triggered by photoactivated hypericin. Our results showed the relocalization of apoptosis-inducing factor (AIF) to the nuclei after hypericin treatment. In addition, we discovered that not only manumycin but also photoactivated hypericin induced the reduction of total Ras protein level. Manumycin decreased the amount of farnesylated Ras, and the combination treatment decreased the amount of both farnesylated and non-farnesylated Ras protein more dramatically. The present findings indicate that the inhibition of Ras processing may be the determining factor for enhancing the antiproliferative and apoptotic effects of combination treatment on HT-29 cells.

Farnesyltransferase Inhibitor Manumycin Targets IL1β-Ras-HIF-1α Axis in Tumor Cells of Diverse Origin

We have recently reported that Ras acts as an intermediate coactivator in IL-1β-mediated hypoxia-inducible factor-1α (HIF-1α) activation in glioblastoma multiforme (GBM). Since HIF-1α plays a crucial role in linking inflammatory and oncogenic pathways, we investigated whether this IL1β-Ras-HIF-1α signaling axis observed in GBM also exists in other tumors of diverse origin under normoxia. Treatment with IL-1β induced Ras in non-GBM cell lines A549 (lung), HeLa (cervical), and HepG2 (liver), and inhibition of Ras activity attenuated HIF-1α activity. Our findings suggest that Ras links IL-1β and HIF-1α in tumors of diverse origin. As we have previously reported that the farnesyltransferase inhibitor manumycin decreases Ras activity in glioma cells, we investigated whether manumycin could regulate IL-1β-mediated HIF-1α activation. Manumycin abrogated IL-1β-induced HIF-1α activation in both glioma and non-glioma tumor cells. In addition, manumycin also decreased IL-1β induced pro-inflammatory responses in tumor cells.

Manumycin inhibits STAT3, telomerase activity, and growth of glioma cells by elevating intracellular reactive oxygen species generation

The poor prognosis of glioblastoma multiforme and lack of effective therapy have necessitated the identification of new treatment strategies. We have previously reported that elevation of oxidative stress induces apoptosis of glioma cells. Because the farnesyltransferase inhibitor manumycin is known to induce reactive oxygen species (ROS) generation, we evaluated the effects of manumycin on glioma cells. Manumycin induced glioma cell apoptosis by elevating ROS generation. Treatment with the ROS inhibitor N-acetylcysteine blocked manumycin-induced apoptosis, caspase-3 activity, and PARP expression, indicating the involvement of increased ROS in the proapoptotic activity of manumycin. This heightened ROS level was accompanied by a concurrent decrease in antioxidants such as superoxide dismutase (SOD-1) and thioredoxin (TRX-1). SOD-1 overexpression protects glioma cells from manumycin-induced apoptosis. In addition, small interfering RNA-mediated knockdown of SOD-1 and TRX-1 expression also increased ROS generation and sensitivity of glioma cells to manumycin-induced cell death. Interestingly, suppressing ROS generation prevented manumycin-induced Ras inhibition. This study reports for the first time that Ras inhibition by manumycin is due to heightened ROS levels. We also report for the first time that manumycin inhibits the phosphorylation of signal transducer and activator of transcription 3 and telomerase activity in a ROS-dependent manner, which plays a crucial role in glioma resistance to apoptosis. In addition manumycin (i) induced the DNA-damage repair response, (ii) affected cell-cycle-regulatory molecules, and (iii) impaired the colony-forming ability of glioma cells in a ROS-dependent manner.

A Farnesyltransferase Inhibitor Manumycin A Induces Oxidative DNA Damage and Apoptosis Via Oxidative Stress in Leukemia Cells.

Farnesyltransferase inhibitors (FTIs) are currently under investigation for the treatment of leukemia. The mechanism of action of FTIs has not been clarified .In this study, we investigated the mechanism of actions of FTI manumycin in acute myeloid leukemia (AML) cells. We found that manumycin A (Manu) had an antineoplastic and apoptotic effect on leukemia cells. Manu significantly increased 8-OhdG, a reliable marker for assessment of oxidative DNA damage, formation. Manu also rapidly increased the phophorylation of H2AX in HL-60 and U937 cells. Methoxyamine, an inhibitor of base excision repair, enhanced the phophorylation of H2AX induced by Manu. Furthermore, Manu could induce phosphated p38 MAPK in HL-60 and U937 cells, and inhibitor of p38 MAPK could rescue leukemia cells from Manu-induced cytotoxicity. Moreover, Quenching of ROS with NAC prevented DNA damage, p38 phosphorylation and apoptosis by Manu. These results suggest the hierarchical position of ROS in oxidative DNA damage and apoptosis pathway. In conclusion, FTI induced oxidative DNA damage by induction of ROS and initiated the DNA damage responses then trigger the apoptosis involved p38 MAPK activation.

Manumycin induces apoptosis in human hepatocellular carcinoma HepG2 cells

Farnesyltransferase inhibitors (FTIs) were developed to prevent Ras processing and thus to be effective agents for the treatment of cancers harbouring mutated ras. In the present study, HepG2 cells underwent internucleosomal DNA fragmentation after treatment with farnesyltransferase inhibitor manumycin (20 microM) for 12 h. Flow cytometric analysis showed that HepG2 cells were accumulated in the G2/M phase of the cell cycle and the number of apoptotic sub-G1 fraction of cells was increased after treatment with manumycin in a time-dependent manner. During the induction of apoptosis, expression of p53 and p21WAF1 was upregulated, phosphorylation of IkappaB-alpha was blocked, caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B were cleaved, and Bcl-2 and Bax protein expression remained unchanged. These results indicated that manumycin induced apoptosis in HepG2 cells. The induction of apoptosis by manumycin involved the upregulation of p53 and p21WAF1, the activation of caspases, and the inhibition of nuclear factor-kappaB (NF-kappaB) pathway. However, Bcl-2 and Bax are not associated with manumycin-mediated apoptosis.

Modulation of RhoA-Rho kinase-mediated Ca2+ sensitization of rabbit myometrium during pregnancy - role of Rnd3.

During pregnancy, the uterus undergoes major functional and structural remodelling. It is well known that during the major part of pregnancy, the myometrium normally remains relatively quiescent but is able to generate powerful contractions at the time of parturition. However, the intracellular molecular events regulating myometrial contractility during pregnancy still remain poorly understood. We applied differential gene expression screening using cDNA array technology to probe myometrium samples from non-pregnant and mid-pregnant (15 days) rabbits. Among the differentially expressed genes, the farnesylated small G-protein of the Rho family, Rnd3, was found to be upregulated (3.6-fold) at mid-pregnancy. Upregulation of Rnd3 was confirmed at the protein level by a 3.4-fold increase in Rnd3 expression in mid-pregnant myometrium. Measurements of contractile properties of beta-escin permeabilized smooth muscle strips revealed that the upregulation of Rnd3 correlated with an inhibition of RhoA-Rho kinase-mediated Ca2+ sensitization at mid-pregnancy. Treatment of muscle strips from mid-pregnant myometrium with the farnesyl-transferase inhibitor manumycin A (10 muM) led to the recovery of RhoA-Rho kinase-dependent Ca2+ sensitization. At late pregnancy (31 days), upregulation of RhoA and Rho kinase expression was associated with an increase in Ca2+ sensitivity of contractile proteins that was inhibited by the Rho kinase inhibitor Y-27632 (10 muM). These data thus demonstrate the time-dependent regulation of the RhoA-Rho kinase-mediated Ca2+ sensitization during the course of pregnancy. The depression of this mechanism at mid-pregnancy followed by its constitutive activation near term is associated with a co-ordinated modulation of Rnd3, RhoA and Rho kinase expression. The RhoA-Rho kinase signalling pathway and its regulators might thus represent potential targets for the development of new treatments for pre-term labour.

Glial Cell Line-Derived Neurotrophic Factor and Target-Dependent Regulation of Large-Conductance KCaChannels in Developing Chick Lumbar Motoneurons

The functional expression of large-conductance Ca2+-activated K+ (K(Ca)) channels in lumbar motoneurons (LMNs) of the developing chick embryo is regulated in part by interactions with striated muscle target tissues. Here we show that the functional expression of K(Ca) channels in LMNs developing in vitro can be stimulated by application of a skeletal muscle extract (MEX) or by coculture with hindlimb myotubes. A similar stimulation of K(Ca) channels in vitro can be produced by the trophic factors glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor but not by neurotrophin (NT)-3 or NT-4. The actions of MEX and hindlimb myotubes are blocked by a GDNF-neutralizing antiserum. Moreover, injection of this same antiserum into the embryonic hindlimb reduced the functional expression of K(Ca) channels in vivo to levels seen in LMNs deprived of interactions with the hindlimb. The effects of GDNF on K(Ca) channel expression in LMNs require 24 hr of continuous exposure to reach maximum and are blocked by the translation inhibitor anisomycin, indicating the need for synthesis of new proteins. GDNF actions are also blocked by the farnesyl transferase inhibitor manumycin, suggesting a role for Ras in the actions of GDNF. Finally, the actions of GDNF are inhibited by PP2, an inhibitor of Src family tyrosine kinases, and by LY29003, an inhibitor of phosphatidylinositol 3 kinases, but not by PD98059, an inhibitor of the Erk signaling cascade. None of these treatments alter expression of voltage-activated Ca2+ channels. Thus, the actions of GDNF on LMN K(Ca) channel expression appear to use a transduction pathway similar to that used for regulation of apoptosis.

Internalin B Activates Nuclear Factor-κB via Ras, Phosphoinositide 3-Kinase, and Akt

Internalin B (InlB), a 630-amino acid protein loosely attached to the surface of Listeria monocytogenes, participates in the entry of the bacterium into mammalian cells. This process requires the activation of phosphoinositide (PI) 3-kinase by InlB. Previously, we demonstrated that InlB activates the transcription factor Nuclear Factor-kappaB in murine J774 macrophage-like cells, an event that also requires PI 3-kinase. Here we have further investigated this phenomenon. InlB activated the small G-protein Ras in J774 cells. Inhibition of Ras with the farnesyltransferase inhibitor manumycin A inhibited NF-kappaB activation and the recruitment of the p85 subunit of PI 3-kinase, implying that Ras is required for PI 3-kinase activation. InlB also activated the PI 3-kinase downstream effector, Akt, as assessed by increased phosphorylation of Akt on serine 473. Transfection of Hep2 cells with dominant negative Ras N17 or dominant negative Akt inhibited the induction of a reporter gene linked to the interleukin-8 promoter by InlB. Furthermore, the Ras inhibitor manumycin A, the PI 3-kinase inhibitor LY294002, and an Akt inhibitor all blocked the induction of interleukin-8 by InlB. Our study is the first report of a bacterial product activating a pathway involving Ras, PI 3-kinase, and Akt, which leads to NF-kappaB activation. This process could be involved in host defense or the inhibition of apoptosis during infection.

The farnesyltransferase inhibitor manumycin A is a novel trypanocide with a complex mode of action including major effects on mitochondria

Eukaryotes modify numerous proteins, including small GTPases of the ras superfamily, with isoprenes as a mechanism for membrane attachment. Inhibition of farnesylation of ras has been successfully exploited to control cell growth, with promise in the clinic for treatment of human tumours. Using an in vitro screen of mammalian farnesyltransferase inhibitors, we have identified manumycin A as potently active against growth of both bloodstream and procyclic forms of Trypanosoma brucei. Other structural classes of farnesyltransferase inhibitors were far less effective. Exposure of T. brucei for brief periods to lethal concentrations of manumycin A resulted in subsequent cell death whilst the concentration required to achieve killing was dependent on serum concentration, suggesting partitioning of manumycin A into hydrophobic cellular sites. Manumycin A did not affect trypanosomal protein and DNA synthesis or cell cycle progression but altered incorporation of prenyl groups into several polypeptides indicating a specific effect on the prenylation without effect on other mevalonate pathway products, most importantly prenyl pyrophosphate levels. Morphological analysis indicated that manumycin A caused significant mitochondrial damage suggesting an additional site of action. Structural analogues of manumycin A containing a quinone were also highly trypanocidal and altered mitochondrial morphology, suggesting interference with electron/proton transport systems. Furthermore, manumycin A also elicited mitochondrial alterations in mammalian cells indicating that the effect is not confined to lower eukaryotes. Manumycin A is well tolerated in vivo but failed to cure experimental trypanosomiasis in mice.