Tyrphostin AG1024 downregulates aryl hydrocarbon receptor (AhR) expression in an IGF1R and IR-independent manner.

The aryl hydrocarbon receptor (AhR) is a receptor-type transcription factor that is crucial for endocrine disruption and carcinogenesis caused by environment chemicals. Previous studies have indicated that certain intracellular signals are involved in AhR activation by their agonists, but the detailed mechanism remains unclear. In this study, we screened for important molecules for AhR activation using SCAD inhibitor kits. Among 164 kinase inhibitors listed in these kits, tyrphostin AG1024, commonly used as an inhibitor of insulin-like growth factor receptor (IGF1R) and insulin receptor (IR), was identified as a potent inhibitor of 3-methylcholanthrene (MC)-mediated AhR activation. We further investigated the mechanism by which AG1024 suppresses MC-mediated AhR activation. AG1024 decreased AhR-dependent luciferase activity, CYP1A1 gene expression, and its protein expression. However, when IGF1R siRNA and IR siRNA were used, AhR activation was slightly increased, in contrast to AG1024 treatment. In addition, AG1024 treatment downregulated the expression of AhR protein but not AhR gene, and decreased both nucleic and cytosolic AhR proteins. Therefore, AG1024 suppressed AhR activation by downregulating AhR protein expression. The molecular target of AG1024 remains unclear, and should be an important target for the regulation of AhR-dependent toxicity.

Developmental defects in zebrafish for classification of EGF pathway inhibitors.

One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairment of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems.

Blockade of EGFR signaling promotes glioma stem-like cell invasiveness by abolishing ID3-mediated inhibition of p27(KIP1) and MMP3 expression.

Aberrant epidermal growth factor receptor (EGFR) signaling is a typical oncogenic signature in glioblastoma. Here, we show that EGFR inhibition in primary glioma stem cells (GSCs) with oncogenic EGFRvIII and EGFRvIII-transduced glioma stem-like cells promotes invasion by decreasing ID3 levels. ID3 suppresses GSC invasiveness by inhibiting p27(KIP1)-RhoA-dependent migration and MMP3 expression. Xenograft and human glioblastoma specimens show that ID3 localizes within glioblastoma cores, whereas p27(KIP1) and MMP3 are predominantly expressed in glioma cells in invasive fronts. Together, our findings show that EGFR inhibition induces GSC invasiveness by abolishing ID3-mediated inhibition of p27(KIP1) and MMP3 expression.

Novel small molecular inhibitors disrupt the JAK/STAT3 and FAK signaling pathways and exhibit a potent antitumor activity in glioma cells.

JAK (Janus kinase)/STAT (signal transducers and activators of transcription) signaling is involved in the regulation of cell growth, differentiation and apoptosis. Constitutive activation of STATs, in particular STAT3, is observed in a large number of human tumors, including gliomas and may contribute to oncogenesis by stimulating cell proliferation and preventing apoptosis, thus it emerges as a promising target for anti-cancer therapy. To investigate the therapeutic potential of blocking STAT3 in glioma cells a set of small synthetic molecules - caffeic acid derivatives, structurally related to AG490 was screened for its ability to inhibit STAT3. Inhibitor 2 (E)-2-cyano-N-[(S)-1-phenylethyl]-3-(pyridin-2-yl)acrylamide was the most effective in inhibition of JAK/STAT3 signaling and at doses ≥ 25 µM significantly reduced the level of phosphorylated JAK1, JAK2 and STAT3 (at Tyr705) and downregulated the expression of known STAT3 targets. In treated cells we observed rapid detachment and rounding of cells associated with reduction of focal adhesion kinase phosphorylation and activity, followed by upregulation of phosphorylated p38, JNK and ERK1/2 levels. Accumulation of cells with fragmented DNA, increases of the cleaved caspase 3 and fragmented PARP levels were detected 24 h after the treatment suggesting ongoing apoptotic cell death. Three human malignant glioblastoma cell lines defective in tumor suppressors TP53 and/or PTEN were susceptible to inhibitor 2 that induced the programmed cell death. Global gene expression profiling revealed modulation of numerous genes in cells treated with inhibitor 2 revealing novel, potential JAK/STAT targets. Our study demonstrates that suitably modified caffeic acid molecules exhibit significant cytotoxic potential toward glioma cells.

Dietary calcium supplementation enhances efficacy but also toxicity of EGFR inhibitor therapy for colon cancer.

The inverse correlation between levels of dietary calcium and colorectal cancer (CRC) incidence has been extensively investigated. However, the impact of supplemental calcium on cancer therapy remains unknown. We used four models of CRC, Caco-2 and HCT116 human cancer cell lines and Apc (Min/+) and azoxymethane carcinogen-induced mouse models, to investigate the impact of a western-style diet low in calcium (0.05%) vs. a similar diet but supplemented with calcium (5%) on therapeutic targeting of the epidermal growth factor receptor (EGFR). We found that calcium supplementation combined with pharmacologic blockade of EGFR results in an additive effect on tumor growth inhibition in all models. Unexpectedly, the combined use of dietary calcium supplementation and EGFR inhibitors also resulted in elevated toxicity suggesting that careful consideration be given when combining dietary supplements with prescribed cancer therapies.

Activation of JAK2/STAT pathway in cerebral cortex after experimental traumatic brain injury of rats.

The janus kinase/signal transducer and activator of transcription (JAK/STAT) is one of the main pathways downstream of cytokine receptors and growth factor receptors by transducing signals from cell surface to the nucleus. In this study, we aimed to survey the role of JAK2/STAT pathway in the progress of TBI. Right parietal cortical contusion in rats was induced by the Feeney free falling model. The activation of JAK2, STAT1 and STAT3 in pericontusional cortex was determined by Western blotting, electrophoretic mobility shift assay (EMSA), immunohistochemistry and immunofluorescence. Moreover, we assessed the neurological recovery (using Neurological Severity Scores (NSS)) of rats under the pretreatment of a JAK2 inhibitor, AG490. Western blotting revealed that expression of p-JAK2, p-STAT1 and p-STAT3 increased immediately, peaked at 3h after TBI and decreased thereafter, and the activation could be inhibited by AG490. Immunohistochemical study showed that JAK2/STAT pathway was activated in both neurons and astrocytes at 3h after TBI. STAT3-specific binding activity was obviously enhanced after TBI and down-regulated after AG490 administration. The higher NSS of TBI+AG490 group revealed a worse behavior recovery when compared with TBI+DMSO group. Our results suggest that the JAK2/STAT pathway is activated in pericontusional cortex of rats, and may be involved in the neurological function recovery after TBI.

Chronic pharmacologic inhibition of EGFR leads to cardiac dysfunction in C57BL/6J mice.

Molecule-targeted therapies like those against the epidermal growth factor receptor (EGFR) are becoming widely used in the oncology clinic. With improvements in treatment efficacy, many cancers are being treated as chronic diseases, with patients having prolonged exposure to several therapies that were previously only given acutely. The consequence of chronic suppression of EGFR activity may lead to unexpected toxicities like altered cardiac physiology, a common organ site for adverse drug effects. To explore this possibility, we treated C57BL/6J (B6) mice with two EGFR small molecule tyrosine kinase inhibitors (TKIs), irreversible EKB-569 and reversible AG-1478, orally for 3 months. In B6 female mice, chronic exposure to both TKIs depressed body weight gain and caused significant changes in left ventricular (LV) wall thickness and cardiac function. No significant differences were observed in heart weight or cardiomyocyte size but histological analysis revealed an increase in fibrosis and in the numbers of TUNEL-positive cells in the hearts from treated female mice. Consistent with histological results, LV apoptotic gene expression was altered, with significant downregulation of the anti-apoptotic gene Bcl2l1. Although there were no significant differences in any of these endpoints in treated male mice, suggesting sex may influence susceptibility to TKI mediated toxicity, the LVs of treated male mice had significant upregulation of Egf, Erbb2 and Nppb over controls. Taken together, these data suggest that chronic dietary exposure to TKIs may result in pathological and physiological changes in the heart.

Conditioning injury-induced spinal axon regeneration requires signal transducer and activator of transcription 3 activation.

Sensory axons in the adult spinal cord do not regenerate after injury. This is essentially because of inhibitory components in the damaged CNS, such as myelin-associated inhibitors and the glial scar. However, if the sciatic nerve is axotomized before injury of the dorsal column, injured axons can regenerate a short distance in the spinal cord. Here, we show that sciatic nerve transection results in time-dependent phosphorylation and activation of the transcription factor, signal transducer and activator of transcription 3 (STAT3), in dorsal root ganglion (DRG) neurons. This effect is specific to peripheral injuries and does not occur when the dorsal column is crushed. Sustained perineural infusion of the Janus kinase 2 (JAK2) inhibitor AG490 to the proximal nerve stump can block STAT3 phosphorylation after sciatic nerve transection and results in reduced growth-associated protein 43 upregulation and compromised neurite outgrowth in vitro. Importantly, in vivo perineural infusion of AG490 also significantly attenuates dorsal column axonal regeneration in the adult spinal cord after a preconditioning sciatic nerve transection. We conclude that STAT3 activation is necessary for increased growth ability of DRG neurons and improved axonal regeneration in the spinal cord after a conditioning injury.

Novel use of zebrafish as a vertebrate model to screen radiation protectors and sensitizers.

PURPOSE: Zebrafish (Danio rerio) embryos provide a unique vertebrate model to screen therapeutic agents easily and rapidly because of their relatively close genetic relationship to humans, ready abundance and accessibility, short embryonal development, and optical clarity. To validate zebrafish embryos as a screen for radiation modifiers, we evaluated the effects of ionizing radiation in combination with a known radioprotector (free radical scavenger Amifostine) or radiosensitizing agent (tyrosine kinase inhibitor AG1478). METHODS AND MATERIALS: Viable zebrafish embryos were exposed to 0-10 Gy single-fraction 250 kVp X-rays with or without either Amifostine (0-4 mM) or AG1478 (0-10 microM) at defined developmental stages from 1-24 h postfertilization (hpf). Embryos were examined for morphologic abnormalities and viability until 144 hpf. RESULTS: Radiation alone produced a time- and dose-dependent perturbation of normal embryonic development and survival with maximal sensitivity at doses > or =4 Gy delivered before 4 hpf. Amifostine markedly attenuated this effect, whereas AG1478 enhanced teratogenicity and lethality, particularly at therapeutically relevant (2-6 Gy) radiation doses. CONCLUSIONS: Collectively, these data validate the use of zebrafish as a vertebrate model to assess the effect of radiation alone or with radiation response modulators. Zebrafish embryos may thus provide a rapid, facile system to screen novel agents ultimately intended for human use in the context of therapeutic or accidental radiation exposure.

Design of hypoxia-targeting protein tyrosine kinase inhibitor using an innovative pharmacophore 2-methylene-4-cyclopentene-1,3-dione.

We review in this report our strategy and tactics for the design of 2-hydroxyarylidene-4-cyclopentene-1,3-diones as protein tyrosine kinase (PTK) inhibitors having low mitochondrial toxicities and/or hypoxia-targeting function. We based our synthetic design on an innovative pharmacophore, 2-methylene-4-cyclopentene-1,3-dione. We first showed the effectiveness of this pharmacophore in the development of 2-methylene-4-cyclopentene-1,3-dione as PTK inhibitor that have lower mitochondrial toxicity than the potent PTK inhibitor tyrphostin AG17. Our results show that the cyclopentenedione-derived TX-1123 is a more potent antitumor tyrphostin and also shows lower mitochondrial toxicity than the malononitrile-derived AG17. The O-methylation product of TX-1123 (TX-1925) retained its tyrphostin-like properties, including mitochondrial toxicity and antitumor activities. However, the methylation product of AG17 (TX-1927) retained its tyrphostin-like antitumor activities, but lost its mitochondrial toxicity. Our comprehensive evaluation of these agents with respect to PTK inhibition, mitochondrial inhibition, antitumor activity, and hepatotoxicity demonstrates that PTK inhibitors TX-1123 and TX-1925 are more promising candidates for antitumor agents than tyrphostin AG17. Secondly, as a further investigation of the promising power of this 4-cyclopentene-1,3-dione as an innovative pharmacophore, we discuss our strategy of development of hypoxia-targeting PTK inhibitor TX-1123 analogues, 2-nitroimidazole-aminomethylenecyclopentenediones, such as TX-2036, for cancer treatment, especially for pancreatic cancers, which have a high level of hypoxia.

Antitumor efficacy of cytotoxic drugs and the monoclonal antibody 806 is enhanced by the EGF receptor inhibitor AG1478.

Blockade of epidermal growth factor receptor (EGFR) signaling with specific inhibitors of the EGFR tyrosine kinase retards cellular proliferation and arrests the growth of tumor xenografts. AG1478, an inhibitor of the EGFR tyrosine kinase, is used in laboratory studies; however, its therapeutic potential has not been elucidated. Therefore, we evaluated an aqueous form of AG1478 for its antitumor activity in mice bearing human xenografts expressing the WT EGFR or a naturally occurring ligand-independent truncation of the EGFR [delta2-7 (de2-7) EGFR or EGFRvIII]. Parenteral administration of soluble AG1478 blocked phosphorylation of the EGFR at the tumor site and inhibited the growth of A431 xenografts that overexpress the WT EGFR and glioma xenografts expressing the de2-7 EGFR. Strikingly, even subtherapeutic doses of AG1478 significantly enhanced the efficacy of cytotoxic drugs, with the combination of AG1478 and temozolomide displaying synergistic antitumor activity against human glioma xenografts. AG1478 was also examined in combination with mAb 806, an anti-EGFR antibody that was raised against the de2-7 EGFR but unexpectedly also binds a subset of the EGFR expressed in cells exhibiting amplification of the EGFR gene. The combination of AG1478 and mAb 806 displayed additive, and in some cases synergistic, antitumor activity against tumor xenografts overexpressing the EGFR. Here, we demonstrate that different classes of inhibitors to the EGFR can have synergistic antitumor activity in vivo. These results establish the antitumor efficacy of the EGFR inhibitor AG1478 and provide a rationale for its clinical evaluation in combination with both chemotherapy and other EGFR therapeutics.

Loss of cell cycle control allows selective microtubule-active drug-induced Bcl-2 phosphorylation and cytotoxicity in autonomous cancer cells.

Lack of selectivity in the killing of tumor and normal cells is a major obstacle in cancer therapy. By inhibiting normal but not autonomous cell growth, we exploited the differences in cell cycle regulation to achieve a selective protection of nonautonomous cells against paclitaxel and other microtubule-active drugs. Tubulin polymerization, a primary effect of paclitaxel, can be dissociated from Bcl-2 phosphorylation and cytotoxicity in HL-60 cells. Growth arrest prevented paclitaxel-induced Bcl-2 phosphorylation and apoptosis without affecting paclitaxel-induced tubulin polymerization. We abrogated the effects of paclitaxel on MCF-10A immortalized breast cells, while preserving its effects on MCF-7 cancer cells. Unlike MCF-7 cells, MCF-10A cells were arrested by epidermal growth factor withdrawal, precluding paclitaxel-induced Bcl-2 phosphorylation. Furthermore, the inhibition of the epidermal growth factor receptor kinase with low doses of AG1478 arrested growth of MCF-10A but not MCF-7 cells. Pretreatment with AG1478 did not affect paclitaxel-induced Bcl-2/Raf-1 phosphorylation in MCF-7 but abrogated such phosphorylation in MCF-10A. Exploitation of growth factor dependency may allow the protection of normal cells from microtubule-active drugs.

Disruption of microtubules in living cells by tyrphostin AG-1714.

Tyrphostin AG-1714 and several related molecules with the general structure of nitro-benzene malononitrile (BMN) disrupt microtubules in a large variety of cultured cells. This process can be inhibited by the stabilization of microtubules with taxol or by pretreatment of the cells with pervanadate, which inhibits tyrosine phosphatases and increases the overall levels of phosphotyrosine in cells. Unlike other microtubule-disrupting drugs such as nocodazole or colchicine, tyrphostin AG-1714 does not interfere with microtubule polymerization or stability in vitro, suggesting that the effect of this tyrphostin on microtubules is indirect. These results imply an involvement of protein tyrosine phosphorylation in the regulation of overall microtubule dynamics. Tyrphostins of AG-1714 type could thus be powerful tools for the identification of such microtubule regulatory pathways.

Effects of the bcr/abl kinase inhibitors AG957 and NSC 680410 on chronic myelogenous leukemia cells in vitro.

The tyrphostin AG957 (NSC 654705) inhibits p210bcr/abl, the transforming kinase responsible for most cases of chronic myelogenous leukemia (CML). The present studies were performed to determine the fate of AG957-treated cells and assess the selectivity of AG957 for CML myeloid progenitors. When K562 cells (derived from a patient with blast crisis CML) were treated with AG957, dose- and time-dependent p210bc/abl down-regulation was followed by mitochondrial release of cytochrome c, activation of caspase-9 and caspase-3, and apoptotic morphological changes. These apoptotic changes were inhibited by transfection with cDNA encoding dominant negative caspase-9 but not dominant-negative FADD or blocking anti-Fas antibodies. In additional experiments, a 24-h AG957 exposure caused dose-dependent inhibition of K562 colony formation in soft agar. To extend these studies to clinical samples of CML, peripheral blood mononuclear cells from 10 chronic phase CML patients and normal controls were assayed for the growth of hematopoietic colonies in vitro in the presence of increasing concentrations of AG957. These assays demonstrated selectivity of AG957 for CML progenitors, with median IC50s (CML versus normal) of 7.3 versus >20 microM AG957 in granulocyte colony-forming cells (P < 0.001), 5.3 versus >20 microM in granulocyte/macrophage colony-forming cells (P < 0.05), and 15.5 versus > 20 microM in erythroid colony-forming cells (P > 0.05). The adamantyl ester of AG957 (NSC 680410) down-regulated p210bcr/abl in K562 cells and inhibited granulocyte colony formation in CML specimens at lower concentrations without enhanced toxicity in normal progenitors. These observations not only demonstrate that AG957-induced p210bcr/abl down-regulation is followed by activation of the cytochrome c/Apaf-1/caspase-9 pathway but also indicate that this class of kinase inhibitor exhibits selectivity worthy of further evaluation.