Withacnistin inhibits recruitment of STAT3 and STAT5 to growth factor and cytokine receptors and induces regression of breast tumours.

BACKGROUND: The binding of STAT3 and STAT5 to growth factor and cytokine receptors such as EGFR and IL-6 receptor gp130 is critical to their activation and ability to contribute to malignant transformation. Therefore, interfering with these biochemical processes could lead to the discovery of novel anticancer agents. METHODS: Co-immunoprecipitation, western blotting, microscopy, DNA binding, invasion, and soft agar assays as well as a mouse model were used to investigate the mechanism by which the natural product Withacnistin (Wit) inhibits STAT 3/5 tyrosine phosphoryaltion and activation. RESULTS: Wit blocks EGF- and IL-6-stimulated binding of STAT3 and STAT5 to EGFR and gp130. Wit inhibits EGF-, PDGF-, IL-6-, IFNß-, and GM-CSF-stimulation of tyrosine phosphorylation of STAT3 and STAT5 but not of EGFR or PDGFR. The inhibition of P-STAT3 and P-STAT5 occurred rapidly, within minutes of Wit treatment and growth factor stimulation. Wit also inhibits STAT3 nuclear translocation, DNA binding, promoter transcriptional activation, and it suppresses the expression levels of STAT3 target genes such as Bcl-xL and Mcl-1. Finally, Wit induces apoptosis, inhibits anchorage-dependent and -independent growth and invasion, and causes breast tumour regression in an ErbB2-driven transgenic mouse model. CONCLUSIONS: These data warrant further development of Wit as a novel anticancer drug for targeting tumours that harbour hyperactivated STAT3 and STAT5.

Identification of novel ROCK inhibitors with anti-migratory and anti-invasive activities.

ROCK1 and ROCK2 mediate important processes such as cell migration, invasion and metastasis, making them good targets for the development of antitumor agents. Recently, using a fragment-based approach and X-ray crystallography, we reported on the design and synthesis of novel Rho-kinase inhibitors (RKIs). Here, we selected a pair of RKIs, the closely related structural analogs RKI-18 (potent; IC50 values of 397 nM (ROCK1) and 349 nM (ROCK2)) and RKI-11 (weak/inactive; IC50 values of 38 µM (ROCK1) and 45 µM (ROCK2)), as chemical probes and determined their effects on cytoskeleton organization, signaling, apoptosis, anchorage-dependent and independent growth, migration and invasion. RKI-18 but not RKI-11 suppresses potently the phosphorylation of the ROCK substrate myosin light chain 2 (MLC2) in intact human breast, lung, colon and prostate cancer cells. Furthermore, RKI-18 is highly selective at decreasing the levels of P-MLC2 over those of P-Akt, P-S6 and P-Erk ½. RKI-18 suppresses ROCK-mediated actin fiber formation, following stimulation with LPA as well as p21-activated kinase (PAK)-mediated lamellipodia and filopodia formation following bradykinin or PDGF stimulation. Furthermore, RKI-18 but not RKI-11 inhibits migration, invasion and anchorage-independent growth of human breast cancer cells. The fact that the active ROCK inhibitor RKI-18, but not the inactive closely related structural analog RKI-11 is effective at suppressing malignant transformation suggests that inhibition of ROCK with RKI-18 results in preventing migration, invasion and anchorage-independent growth. The potential of this class of RKIs as anti-tumor agents warrants further advanced preclinical studies.

The Akt activation inhibitor TCN-P inhibits Akt phosphorylation by binding to the PH domain of Akt and blocking its recruitment to the plasma membrane.

Persistently hyperphosphorylated Akt contributes to human oncogenesis and resistance to therapy. Triciribine (TCN) phosphate (TCN-P), the active metabolite of the Akt phosphorylation inhibitor TCN, is in clinical trials, but the mechanism by which TCN-P inhibits Akt phosphorylation is unknown. Here we show that in vitro, TCN-P inhibits neither Akt activity nor the phosphorylation of Akt S473 and T308 by mammalian target of rapamycin or phosphoinositide-dependent kinase 1. However, in intact cells, TCN inhibits EGF-stimulated Akt recruitment to the plasma membrane and phosphorylation of Akt. Surface plasmon resonance shows that TCN, but not TCN, binds Akt-derived pleckstrin homology (PH) domain (K(D): 690 nM). Furthermore, nuclear magnetic resonance spectroscopy shows that TCN-P, but not TCN, binds to the PH domain in the vicinity of the PIP3-binding pocket. Finally, constitutively active Akt mutants, Akt1-T308D/S473D and myr-Akt1, but not the transforming mutant Akt1-E17K, are resistant to TCN and rescue from its inhibition of proliferation and induction of apoptosis. Thus, the results of our studies indicate that TCN-P binds to the PH domain of Akt and blocks its recruitment to the membrane, and that the subsequent inhibition of Akt phosphorylation contributes to TCN-P antiproliferative and proapoptotic activities, suggesting that this drug may be beneficial to patients whose tumors express persistently phosphorylated Akt.

A chemical biology approach identifies a beta-2 adrenergic receptor agonist that causes human tumor regression by blocking the Raf-1/Mek-1/Erk1/2 pathway.

A chemical biology approach identifies a beta 2 adrenergic receptor (beta2AR) agonist ARA-211 (Pirbuterol), which causes apoptosis and human tumor regression in animal models. beta2AR stimulation of cAMP formation and protein kinase A (PKA) activation leads to Raf-1 (but not B-Raf) kinase inactivation, inhibition of Mek-1 kinase and decreased phospho-extracellular signal-regulated kinase (Erk)1/2 levels. ARA-211 inhibition of the Raf/Mek/Erk1/2 pathway is mediated by PKA and not exchange protein activated by cAMP (EPAC). ARA-211 is selective and suppresses P-Erk1/2 but not P-JNK, P-p38, P-Akt or P-STAT3 levels. beta2AR stimulation results in inhibition of anchorage-dependent and -independent growth, induction of apoptosis in vitro and tumor regression in vivo. beta2AR antagonists and constitutively active Mek-1 rescue from the effects of ARA-211, demonstrating that beta2AR stimulation and Mek kinase inhibition are required for ARA-211 antitumor activity. Furthermore, suppression of growth occurs only in human tumors where ARA-211 induces cAMP formation and decreases P-Erk1/2 levels. Thus, beta2AR stimulation results in significant suppression of malignant transformation in cancers where it blocks the Raf-1/Mek-1/Erk1/2 pathway by a cAMP-dependent activation of PKA but not EPAC.

Farnesyltransferase and geranylgeranyltransferase I inhibitors upregulate RhoB expression by HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter.

Recently, we have shown that RhoB suppresses EGFR-, ErbB2-, Ras- and Akt-mediated malignant transformation and metastasis. In this paper, we demonstrate that the novel antitumor agents farnesyltransferase inhibitors (FTIs) and geranylgeranyltransferase I inhibitors (GGTIs) upregulate RhoB expression in a wide spectrum of human cancer cells including those from pancreatic, breast, lung, colon, bladder and brain cancers. RhoB induction by FTI-277 and GGTI-298 occurs at the transcriptional level and is blocked by actinomycin D. Reverse transcription-PCR experiments documented that the increase in RhoB protein levels is due to an increase in RhoB transcription. Furthermore, treatment with FTIs and GGTIs of cancer cells results in HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter. Thus, promoter acetylation is a novel mechanism by which RhoB expression levels are regulated following treatment with the anticancer agents FTIs and GGTIs.

Natural phosphate doped with KI in HMDS: a mild and efficient reagent for alkylation and glycosylation of nucleobases.

Several D-ribonucleosides are prepared from 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranoside and trimethylsilylated nucleobases under mild conditions by using natural phosphate doped with KI as catalyst.

Removal of lead from aqueous solutions by activated phosphate.

The potential of using activated phosphate as a new adsorbent for the removal of Pb from aqueous solutions was investigated. The kinetic of lead adsorption and the adsorption process were compared for natural phosphate (NP) and activated phosphate (AP). The results indicate that equilibrium was established in about 1h for NP and 3 h for AP. The effect of the pH was examined in the range 2-6. The maximum removal obtained is between two and three for NP and between three and four for AP. The maximum adsorption capacities at 25 degrees C are 155.04 and 115.34 mg/g for AP and NP, respectively. The effect of temperature has been carried out at 25, 35 and 45 degrees C. The data obtained from adsorption isotherms of lead at different temperatures fit to linear form of Langmuir adsorption equation. The thermodynamic parameters such as enthalpy (DeltaH), free energy (DeltaG) and entropy (DeltaS) were calculated. They show that adsorption of lead on NP and AP is an endothermic process more effective at high temperatures. These results show that AP is a good adsorbent for heavy metals from aqueous solutions and could be used as a purifier for water and wastewater.

Removal of heavy metal ions from water by using calcined phosphate as a new adsorbent.

Calcined phosphate (CP) has been employed in our laboratories as a heterogeneous catalyst in a variety of reactions. In this study, CP was evaluated as a new product for removal of heavy metals from aqueous solution. Removal of Pb2+, Cu2+, and Zn2+ on the CP was investigated in batch experiments. The kinetic of lead on CP adsorption efficiency and adsorption process were evaluated and analysed using the theories of Langmuir and Freundlich. The influence of pH was studied. The adsorption capacity obtained at pH 5 were 85.6, 29.8, and 20.6 mg g(-1) for Pb2+, Cu2+ and Zn2+, respectively. We hypothesize at pH 2 and 3, the dissolution of CP and precipitation of a fluoropyromorphite for lead and the formation of solid-solution type fluorapatite for copper. The results obtained show that CP is a good adsorbent for these toxic heavy metals. The abundance of natural phosphate, its low price and non-aggressive nature towards the environment are advantage for its utilisation in point of view of wastewater and wastes clean up.

Doped natural phosphate: a new and environmentally friendly catalyst in nucleoside synthesis.

Doped natural phosphate is used as acidic or basic catalyst in nucleoside and acyclonucleoside synthesis. Some examples are given.

Building blocks for polyamide nucleic acids: facile synthesis using potassium fluoride doped natural phosphate as basic catalyst.

Potassium fluoride doped natural phosphate, inexpensive and environmentally friendly catalyst, is shown to be an efficient basic catalyst for the N1/N9 alkylation of different nucleobases as synthons for PNAs.

The farnesyl transferase inhibitor, FTI-277, inhibits growth and induces apoptosis in drug-resistant myeloma tumor cells.

Mutations of the ras gene are among the most commonly identified transforming events in human cancers, including multiple myeloma. Farnesyltransferase inhibitors (FTI) were developed to prevent Ras processing and induce cancer cell death. Several FTIs are in phase II and one is in phase III clinical trials. Preclinically, most of the focus has been on solid tumors, and the effects of FTIs in multiple myeloma have not been investigated. In this study we examined the cytotoxic activity and inhibition of Ras processing in three myeloma cell lines with differing Ras mutation status. H929 cells with activated N-Ras were more sensitive to FTI-277 treatment than 8226 and U266 cells with activated K-Ras or wild-type Ras, respectively. A combination of FTI-277 and a geranylgeranyltransferase I inhibitor (GGTI)-2166 inhibited K-Ras processing and enhanced cell death in 8226 cells. U266 cells and Bcl-x(L) transfectants were equally sensitive to FTI-277 treatment. Similarly, 8226 cells selected for resistance to various chemotherapeutic agents, which resulted in either P-glycoprotein overexpression, altered topoisomerase II activity, or elevated glutathione levels, were equally sensitive to FTI-277. These preclinical studies suggest that prenylation inhibitors may represent new therapeutic agents for the treatment of refractory or drug-resistant multiple myeloma.

QSAR for anti-HIV activity of HEPT derivatives.

QSARs were derived for 103 analogues of 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT), a potent inhibitor of the HIV-1 reverse transcriptase (RT). The activity of these compounds was investigated by means of multiple linear regression (MLR) and artificial neural network (ANN) techniques. Considering the relevant descriptors obtained from the MLR, a correlation coefficient of 0.92 (n = 95) was obtained with a 4-5-1 ANN model. The contribution of each descriptor to the structure-activity relationships was evaluated. The results showed that the anti-HIV activity of HEPT derivatives was strongly dependent on hydrophobic character and also steric factors of substituents.

The farnesyltransferase inhibitor, FTI-2153, inhibits bipolar spindle formation during mitosis independently of transformation and Ras and p53 mutation status.

Recently, we have shown that the farnesyltransferase inhibitor FTI-2153 induces accumulation of two human lung cancer cell lines in mitosis by inhibiting bipolar spindle formation during prometaphase. Here we investigate whether this mitotic arrest depends on transformation, Ras and/or p53 mutation status. Using DAPI staining (DNA) and immunocytochemistry (microtubules), we demonstrate that in normal primary foreskin fibroblasts (HFF), as well as in several cancer cell lines of different origins including human ovarian (OVCAR3), lung (A-549 and Calu-1) and fibrosarcoma (HT1080), FTI-2153 inhibits bipolar spindle formation and induces a rosette morphology with a monopolar spindle surrounded by chromosomes. In both malignant cancer cell lines and normal primary fibroblasts, the percentage of prometaphase cells with bipolar spindles decreases from 67-92% in control cells to 2-28% in FTI-2153 treated cells. This inhibition of bipolar spindle formation correlates with an accumulation of cells in prometaphase. The ability of FTI-2153 to inhibit bipolar spindle formation is not dependent on p53 mutation status since both wild-type (HFF, HT1080 and A-549) and mutant (Calu-1 and OVCAR3) p53 cells were equally affected. Similarly, both wild-type (HFF and OVCAR3) and mutant (HT1080, Calu-1 and A-549) Ras cells accumulate monopolar spindles following treatment with FTI-2153. However, two cell lines, NIH3T3 (WT Ras and WT p53) and the human bladder cancer cell line, T-24 (mutant H-Ras and mutant p53) are highly resistant to FTI-2153 inhibition of bipolar spindle formation. Finally, the ability of FTI-2153 to inhibit tumor cell proliferation does not correlate with inhibition of bipolar spindle formation. Taken together these results demonstrate that the ability of FTI-2153 to inhibit bipolar spindle formation and accumulate cells in mitosis is not dependent on transformation, Ras or p53 mutation status. Furthermore, in some cell lines, FTIs inhibit growth by mechanisms other than interfering with the prophase/metaphase traverse.

Phosphotyrosyl peptides block Stat3-mediated DNA binding activity, gene regulation, and cell transformation.

Signal transducers and activators of transcription (STATs) comprise a family of cytoplasmic signaling proteins that participates in normal cellular responses to cytokines and growth factors. Frequently, however, constitutive activation of certain STAT family members, particularly Stat3, has accompanied a wide variety of human malignancies. To identify small molecule inhibitors of Stat3, we investigated the ability of the Stat3 SH2 domain-binding peptide, PY*LKTK (where Y* represents phosphotyrosine), to disrupt Stat3 activity in vitro. The presence of PY*LKTK, but not PYLKTK or PFLKTK, in nuclear extracts results in significant reduction in the levels of DNA binding activities of Stat3, to a lesser extent of Stat1, and with no effect on that of Stat5. Analyses of alanine scanning mutagenesis and deletion derivatives of PY*LKTK reveal that the Leu residue at the Y+1 position and a substituent at the Y-1 position (but not necessarily Pro) are essential for the disruption of active Stat3, thereby mapping the minimum active sequence to the tripeptide, XY*L. Studies involving bead-coupled PY*LKTK peptide demonstrate that this phosphopeptide directly complexes with Stat3 monomers in vitro, suggesting that PY*LKTK disrupts Stat3:Stat3 dimers. As evidence for the functional importance of peptide-directed inhibition of Stat3, PY*LKTK-mts (mts, membrane translocating sequence) selectively inhibits constitutive and ligand-induced Stat3 activation in vivo. Furthermore, PY*LKTK-mts suppresses transformation by the Src oncoprotein, which has been shown previously to require constitutive Stat3 activation. Altogether, we have identified a minimal peptide that inhibits Stat3 signaling and provides the conceptual basis for use of this peptide as a lead for novel peptidomimetic drug design.

Signaling events required for transforming growth factor-beta stimulation of connective tissue growth factor expression by cultured human lung fibroblasts.

It is possible that many of the fibrogenic effects of transforming growth factor-beta (TGF-beta) are mediated by connective tissue growth factor (CTGF). In the present work, we show that TGF-beta1 produces a 5- to 6-fold increase in CTGF expression by cultured human lung fibroblasts that is due mainly to increased transcription. The half-life of CTGF mRNA is 1.96 h, consistent with its role as a cytokine. In addition to requiring Smad activity, based upon the effects of specific inhibitors, the TGF-beta intracellular signaling pathway requires the activity of a phosphatidylcholine-specific phospholipase C, a protein kinase C, and one or more tyrosine kinases. It is also likely that the pathway requires a member of the Ras superfamily of small GTPases, but not trimeric G proteins. Pharmacologic inhibition of TGF-beta stimulation of CTGF expression may be an effective therapeutic approach to a variety of undesirable fibrotic reactions.

Ras and RhoA suppress whereas RhoB enhances cytokine-induced transcription of nitric oxide synthase-2 in human normal liver AKN-1 cells and lung cancer A-549 cells.

While both nitric oxide synthase-2 (NOS-2) and low molecular weight GTPases, such as Ras and Rho, have been implicated in malignant transformation, the cross talk between these important proteins is ill understood. In this study we examined the ability of H-Ras, RhoA, RhoB and Rac1 to modulate cytokine-induced NOS2. In the normal human liver AKN-1 cell line and in the human non-small cell lung carcinoma cell line, A-549, the ability of the cytokines (INF-gamma, IL-1beta and TNF-alpha) to activate NOS-2 was blocked by activated L61-H-Ras whereas dominant negative N17-H-Ras enhanced NOS-2 activation. Consistent with this dominant negative Erk2 as well as a MEK inhibitor also enhanced cytokine activation of NOS-2. Furthermore, activated L63-RhoA blocked whereas activated V14-RhoB enhanced cytokine NOS-2 activation. Activated I115-Racl did not affect NOS-2 activation. These results demonstrate that the Ras/Erk and the Ras/RhoA pathways negatively regulate whereas RhoB enhances cytokine-induced NOS-2. This is the first demonstration that genes that promote malignant transformation such as Ras and RhoA inhibit, whereas genes with tumor suppressor activity such as RhoB enhance NOS2 induction.

Signal transduction pathways of IL-1beta-mediated iNOS in pulmonary vascular smooth muscle cells.

Interleukin (IL)-1beta is an important early mediator of inflammation in pulmonary artery smooth muscle cells. We previously reported that a geranylgeranyltransferase inhibitor elevated basal levels of inducible nitric oxide synthase (iNOS) and enhanced IL-1beta-mediated induction, suggesting that Rac or Rho small G proteins are candidates for antagonism of such induction. In this study, overexpression of constitutively active Rac1 or its dominant negative mutant did not affect IL-1beta induction of iNOS. Alternatively, treatment with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho, was associated with superinduction of iNOS, suggesting an inhibitory role for Rho. IL-1beta activated the three mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2, c-Jun NH2-terminal kinase/stress-activated protein kinase, and p38) and the Janus kinase (JAK)-signal transducer and activator of transcription pathways. The former two pathways were not associated with IL-1beta-mediated iNOS induction, whereas the latter two appeared to have inhibitory roles in iNOS expression. These data suggest that a broad intracellular signaling response to IL-1beta in rat pulmonary artery smooth muscle cells results in elevated levels of iNOS that is opposed by the geranylgeranylated small G protein Rho as well as the p38 and JAK2 pathways.

v-Src transformation is mediated through farnesylated proteins.

Src is an oncoprotein which has been implicated in a number of human malignancies in which it has been shown to be overexpressed and highly activated. The precise mechanism of Src transformation, however, is still poorly understood. We hypothesized that Ras and other farnesylated proteins may mediate Src transformation. To test this hypothesis, v-Src-transfected rat fibroblasts (3Y1) were treated every 72 h with a 15 microM concentration of a farnesyl-transferase inhibitor (FTI). At 2 weeks, a focus formation assay was performed to assess transformation potential. Untreated and FTI-treated v-Src-transfected 3Y1 cells formed a mean of 39 (+/-2.6) and 29.8 (+/-2.9) foci per well, respectively. This 24% decrease was judged to be statistically significant (P = 0.02). Moreover, foci (>90%) in the FTI-treated wells were also consistently smaller than foci in the untreated wells. Western blots with antibody directed toward H-Ras confirmed complete inhibition of Ras farnesylation in the treated cell lines. The specificity of this inhibition was verified by Western blot using antibody specific for Rap1A. The transforming potential of v-Src is inhibited, but not eliminated by FTI treatment. This suggests that v-Src transformation is mediated in part by farnesylated proteins, one of which may be Ras.

Peptidomimetic inhibitors of protein farnesyltransferase show potent antimalarial activity.

Malaria continues to represent a very serious health problem in the tropics. The current methods of clinical treatment are showing deficiencies due to the increased incidence of resistance in the parasite. In the present paper we report the design, synthesis, and evaluation of potential antimalarial agents against a novel target, protein farnesyltransferase. We show that the most potent compounds are active against Plasmodium falciparum in vitro at submicromolar concentrations.

CEP1612, a dipeptidyl proteasome inhibitor, induces p21WAF1 and p27KIP1 expression and apoptosis and inhibits the growth of the human lung adenocarcinoma A-549 in nude mice.

The ubiquitin proteasome system is responsible for the proteolysis of important cell cycle and apoptosis-regulatory proteins. In this paper we report that the dipeptidyl proteasome inhibitor, phthalimide-(CH2)8CH-(cyclopentyl) CO-Arg(NO2)-Leu-H (CEP1612), induces apoptosis and inhibits tumor growth of the human lung cancer cell line A-549 in an in vivo model. In cultured A-549 cells, CEP1612 treatment results in accumulation of two proteasome natural substrates, the cyclin-dependent kinase inhibitors p21WAF1 and p27KIP1, indicating its ability to inhibit proteasome activity in intact cells. Furthermore, CEP1612 induces apoptosis as evident by caspase-3 activation and poly(ADP-ribose) polymerase cleavage. Treatment of A-549 tumor-bearing nude mice with CEP1612 (10 mg/kg/day, i.p. for 31 days) resulted in massive induction of apoptosis and significant (68%; P < 0.05) tumor growth inhibition, as shown by terminal deoxynucleotidyltransferase-mediated UTP end labeling. Furthermore, immunostaining of tumor specimens demonstrated in vivo accumulation of p21WAF1 and p27KIP1 after CEP1612 treatment. The results suggest that CEP1612 is a promising candidate for further development as an anticancer drug and demonstrate the feasibility of using proteasome inhibitors as novel antitumor agents.