A small-molecule enhancer of signal transducer and activator of transcription 1 transcriptional activity accentuates the antiproliferative effects of IFN-gamma in human cancer cells.

The transcription factor signal transducer and activator of transcription (STAT) 1 can mediate antiproliferative and proapoptotic effects in cancer cells, and a number of mechanisms have been found whereby STAT1 signaling is attenuated in tumors thereby increasing their malignant behavior. Thus, enhancing gene transcription mediated by STAT1 may be an effective approach to cancer therapy. A high-throughput screen was developed to identify molecules that could enhance STAT1-dependent gene expression. Through this approach, it was found that 2-(1,8-naphthyridin-2-yl)phenol (2-NP) caused a 2-fold increase in STAT1-dependent reporter gene expression compared with that seen with maximally effective concentrations of IFN-gamma alone. This effect was specific to STAT1 because 2-NP had no effect on unrelated transcription factors such as nuclear factor (NF) kappaB or the highly homologous transcription factor STAT3. STAT1-dependent gene activation was enhanced by this compound in a variety of human and murine cell lines and was independent of the stimulus used. Furthermore, 2-NP enhanced the expression of the bona fide endogenous STAT1 target gene interferon regulatory factor 1. 2-NP increased the duration of STAT1 tyrosine phosphorylation in response to IFN-gamma, and this may underlie its enhancement of STAT1-dependent transcription. Reflecting the fact that STAT1 can exert tumor-suppressive effects, 2-NP enhanced the ability of IFN-gamma to inhibit the proliferation of human breast cancer and fibrosarcoma cells. Tumor cells lacking STAT1 were unaffected by either IFN-gamma or 2-NP. These findings indicate that enhancement of STAT1 transcriptional activity may have utility in anticancer therapies, and that cell-based screens for modulators of transcription factor function can be a useful approach for drug discovery.

Carbazole is a naturally occurring inhibitor of angiogenesis and inflammation isolated from antipsoriatic coal tar.

Coal tar is one of the oldest and an effective treatment for psoriasis. Coal tar has been directly applied to the skin, or used in combination with UV light as part of the Goeckerman treatment. The use of coal tar has caused long-term remissions in psoriasis, but has fallen out of favor because the treatment requires hospitalization and coal tar is poorly acceptable aesthetically to patients. Thus, determining the active antipsoriatic component of coal tar is of considerable therapeutic interest. We fractionated coal tar into its components, and tested them using the SVR angiogenesis inhibitor assay. Treatment of SVR endothelial cells with coal tar fractions resulted in the isolation of a single fraction with antiangiogenic activity. The active antiangiogenic compound in coal tar is carbazole. In addition to antiangiogenic activity, carbazole inhibited the production of inflammatory IL-15 by human mononuclear cells. IL-15 is elevated in psoriasis and is thought to contribute to psoriatic inflammation. Carbazole treatment also reduced activity of inducible nitric oxide synthase (iNOS), which is proinflammatory and elevated in psoriasis. The effect of carbazole on upstream pathways in human psoriasis was determined, and carbazole was shown to inhibit signal transducer and activator of transcription (stat)3-mediated transcription, which has been shown to be relevant in human psoriasis. IL-15, iNOS, and stat3 activation require the activation of the small GTPase rac for optimal activity. Carbazole was found to inhibit rac activation as a mechanism for its inhibition of downstream inflammatory and angiogenic pathways. Given its antiangiogenic and anti-inflammatory activities, carbazole is likely a major component of the antipsoriatic activity of coal tar. Carbazole and derivatives may be useful in the therapy of human psoriasis.

Signal transducer and activator of transcription 1 activation in endothelial cells is a negative regulator of angiogenesis.

To determine the role of the transcription factor signal transducer and activator of transcription (STAT) 1 on endothelial cell function, human umbilical vein endothelial cells (HUVEC) were treated with IFN-gamma, a potent activator of STAT1. IFN-gamma inhibited cell growth and tube formation of HUVECs. Although the potent proangiogenic protein vascular endothelial growth factor (VEGF) stimulated cell growth and tube formation, IFN-gamma could suppress these effects of VEGF. Transfection of HUVECs with short interfering RNA targeting STAT1 abrogated IFN-gamma-induced inhibition of HUVEC growth and tube formation, and suppressed the inhibition of VEGF-induced tube formation by IFN-gamma, indicating that STAT1 is critical for this process. IFN-gamma blocks the biological activity of VEGF through inhibition of genes necessary for the VEGF response, including angiopoietin-2, urokinase plasminogen activator, tissue inhibitor of matrix metalloproteinase-1, cyclooxygenase-2, and VEGF receptor 2. To extend these findings in vivo, the role of STAT1 in angiogenesis was examined in STAT1-deficient mice using the Matrigel in vivo angiogenesis assay. Substantial cellular infiltration and formation of vascular structures occurred in STAT1-/- mice compared with wild-type controls. These data indicate that STAT1 plays a key role in the inhibition of angiogenesis through its action within endothelial cells, and exploiting this process may be useful in treating cancers and vascular tumors.

A recombinant IL-4-Pseudomonas exotoxin inhibits protein synthesis and overcomes apoptosis resistance in human CLL B cells.

We have determined that CLL B cells consistently express type 3 membrane receptors for the Th2-derived cytokine IL-4 (IL-4R). Furthermore, when added to CLL B cells, IL-4 induces increased apoptosis resistance, increased protein synthesis in CLL B cells and rapid onset activation of STAT1, STAT5 and STAT6. Since the IL-4-IL-4R pathway is intact in CLL B cells and is related to apoptosis resistance, we considered whether we could target this pathway. A recombinant IL-4 Pseudomonas exotoxin fusion protein (IL-4 PE), known to bind to IL-4R, was incubated with CLL B cells. IL-4 PE (10 ng/ml) cultured with CLL B cells resulted in an increase of apoptosis/death from mean levels of 46.6+/-7.0 of non-exposed cells to 69+/-8.6 (n=6). By measuring in vitro protein synthesis, two predominant patterns of sensitivity were observed. In one, CLL B cell clones (n=4) were found to be extremely sensitive to IL-4 PE (IC50's range=6-25 ng/ml). In the second, low concentrations of IL-4 PE induced agonist activity while increasing concentrations induced cytotoxicity in 6 of 21 patient-derived cells. These studies suggest that the IL-4R, on B-CLL cells, can serve as a unique molecular target for directing cytotoxic agents in the therapy of B-CLL.

The natural product honokiol induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells.

B-cell chronic lymphocytic leukemia (B-CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Honokiol is a natural product known to possess potent antineoplastic and antiangiogenic properties. We examined whether honokiol can overcome apoptotic resistance in primary tumor cells derived from B-CLL patients. Honokiol induced caspase-dependent cell death in all of the B-CLL cells examined and was more toxic toward B-CLL cells than to normal mononuclear cells, suggesting greater susceptibility of the malignant cells. Honokiol-induced apoptosis was characterized by the activation of caspase-3, -8, and -9 and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP). Exposure of B-CLL cells to honokiol resulted in up-regulation of Bcl2-associated protein (Bax) and down-regulation of the expression of the key survival protein myeloid-cell leukemia sequence 1 (Mcl-1), which is associated with response to treatment in B-CLL patients. In addition, B-CLL cells pretreated with interleukin-4 (IL-4), a cytokine known to support B-CLL survival, underwent apoptosis when subsequently incubated with honokiol, indicating that honokiol could also overcome the prosurvival effects of IL-4. Furthermore, honokiol enhanced cytotoxicity induced by fludarabine, cladribine, or chlorambucil. These data indicate that honokiol is a potent inducer of apoptosis in B-CLL cells and should be examined for further clinical application either as a single agent or in combination with other anticancer agents.

Oral fludarabine has significant activity in patients with previously untreated chronic lymphocytic leukemia, and leads to increased STAT1 levels in vivo.

Fifteen patients with previously untreated chronic lymphocytic leukemia (CLL) were treated with oral fludarabine. Toxicities were mainly hematologic, and the response rate was 80%. To assess the effect of fludarabine on the transcription factor STAT1, blood samples obtained on study entry were treated in vitro with fludarabine for 24 h, and the majority of samples displayed an expected decrease in STAT1. To determine whether similar changes occurred in vivo, we developed a flow cytometric assay to quantitate STAT1 levels. On completion of fludarabine cycle 1, CLL cells showed increased STAT1 in the majority of patients, in contrast to the in vitro findings. This may reflect a survival advantage for cells that express high levels of STAT1. In conclusion, oral fludarabine is highly active and merits further investigation in previously untreated patients with CLL. Larger studies are indicated to determine optimal timing of STAT1 assessment, and if changes in STAT1 represent an in vivo indicator of response to purine analog therapy.

STAT1 mediates differentiation of chronic lymphocytic leukemia cells in response to Bryostatin 1.

Bryostatin 1 is known to exhibit in vitro and in vivo activity against chronic lymphocytic leukemia (CLL) cells by inducing their further maturation into plasma-like cells. Signal transducer and activator of transcription (STAT) proteins play a central role in B-lymphocyte growth and function and are aberrantly phosphorylated on serine residues in CLL cells. To determine whether STAT transcription factors are important in Bryostatin 1-induced differentiation of CLL cells, primary CLL cells were examined for signaling events following exposure to Bryostatin 1 in vitro. Western analysis and electrophoretic mobility shift assays revealed that Bryostatin 1 induced tyrosine phosphorylation and DNA binding of STAT1, yet there was no effect on constitutive serine phosphorylation of STAT1. Bryostatin 1-induced STAT1 activation occurred in a manner that was dependent on protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Janus tyrosine kinase (JAK) activation. Evidence indicates that Bryostatin 1 induces STAT1 activation through an interferon gamma (IFN gamma) autocrine loop. However, STAT1 activation by IFN gamma stimulation alone was not sufficient to induce differentiation. This insufficiency is due to the broader effect on gene expression caused by Bryostatin 1 compared with IFN gamma, as demonstrated by microarray analysis. Both up-regulation of CD22 expression and immunoglobulin M (IgM) production, markers of CLL differentiation, were inhibited by a decoy oligonucleotide for STAT1, indicating that STAT1 is necessary for Bryostatin 1-induced differentiation of CLL cells. This study implicates STAT transcription factors as important mediators of Bryostatin 1-induced differentiation of CLL cells and could possibly lead to improved therapeutic approaches for the treatment of CLL.

In vivo activation of signal transducer and activator of transcription 1 after CD154 gene therapy for chronic lymphocytic leukemia is associated with clinical and immunologic response.

PURPOSE: Signal transducer and activator of transcription (STAT) proteins are important regulators of physiological stimuli in lymphocytes. Biological therapies directed at lymphocytic malignancies such as chronic lymphocytic leukemia (CLL) may be mediated by these transcription factors. One such approach, CD154 (CD40-ligand) gene therapy, involves expressing CD154 on malignant B cells from CLL patients by transduction with an adenovirus vector after which the cells are reinfused into the patients. To determine the intracellular signaling pathways that underlie the clinical and immunological responses observed in patients from a Phase I study of CD154 gene therapy, CLL cells from these patients were examined for changes in STAT signaling events. EXPERIMENTAL DESIGN: CLL cells from patients who underwent CD154 gene therapy were analyzed for changes in STAT signaling by Western blot analysis and electrophoretic mobility shift assay. Activation of STAT1 was correlated with patient response to therapy. RESULTS: Tyrosine phosphorylation of STAT1 was detected in the nontransduced CLL cells in 9 of 11 patients 24 h after infusion, but not before. Activation of STAT1 was associated with clinical response, as measured by decreased absolute lymphocyte count, and immunological response, as measured by elevated plasma levels of IFN-gamma. CONCLUSION: This study indicates that STAT signaling may be an important mediator of biological treatments, such as CD154 gene therapy, and that early STAT1 activation may predict response to this novel treatment.

Sustained complete remission of CLL associated with the use of a Chinese herbal extract: case report and mechanistic analysis.

We report the case of a man with chronic lymphocytic leukemia (CLL) who, in the absence of cytotoxic chemotherapy, began taking a Chinese herbal extract. Shortly thereafter, he experienced a steady decline of his lymphocytosis and adenopathy, and he remains in remission now over 10 years later. The herbal extract inhibited the survival of primary CLL cells under in vitro culture conditions. However, it did not inhibit the activation of Akt or MAP kinase, nor did it inhibit the serine phosphorylation of STAT1. Thus, through an as yet unknown mechanism, this extract appears to exert pro-apoptotic effects in CLL.

Ectopic expression of CXCR5/BLR1 accelerates retinoic acid- and vitamin D(3)-induced monocytic differentiation of U937 cells.

The product of the blr1 gene is a CXC chemokine receptor (CXCR5) that regulates B lymphocyte migration and has been implicated in myelomonocytic differentiation. The U937 human leukemia cell line was used to study the role of blr1 in retinoic acid-regulated monocytic leukemia cell growth and differentiation. blr1 mRNA expression was induced within 12 hr by retinoic acid in U937 cells. To determine whether the early induction of blr1 might regulate inducible monocytic cell differentiation, U937 cells were stably transfected with blr1 (U937/blr1 cells). Ectopic expression of blr1 caused no significant cell cycle or differentiation changes, but caused the U937/blr1 cells to differentiate faster when treated with either retinoic acid or 1alpha,25-dihydroxyvitamin D(3). Treated with retinoic acid, U937/blr1 cells showed a greater increase in the percentage of CD11b expressing cells than vector control cells. Retinoic acid also induced a higher percentage of functionally differentiated blr1 transfectants as assessed by nitroblue tetrazolium reduction. U937/blr1 cells underwent moderate growth inhibition on treatment with retinoic acid. Similar results occurred with 1alpha,25-dihydroxyvitamin D(3). Because blr1 was induced early during cell differentiation and because its overexpression accelerated monocytic differentiation, it may be important for signals controlling cell differentiation.