Thymoquinone Induces Downregulation of BCR-ABL/JAK/STAT Pathway and Apoptosis in K562 Leukemia Cells.

OBJECTIVE: BCR ABL oncogene encodes the BCR-ABL chimeric protein, which is a constitutively activated non-receptor tyrosine kinase. The BCR-ABL oncoprotein is a key molecular basis for the pathogenesis of chronic myeloid leukemia (CML) via activation of several downstream signaling pathways including JAK/STAT pathway. Development of leukemia involves constitutive activation of signaling molecules including, JAK2, STAT3, STAT5A and STAT5B. Thymoquinone (TQ) is a bioactive constituent of Nigella sativa that has shown anticancer properties in various cancers. The present study aimed to evaluate the effect of TQ on the expression of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes and their consequences on the cell proliferation and apoptosis in K562 CML cells. METHODS: BCR-ABL positive K562 CML cells were treated with TQ. Cytotoxicity was determined by Trypan blue exclusion assay. Apoptosis assay was performed by annexin V-FITC/PI staining assay and analyzed by flow cytometry. Transcription levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Protein levels of JAK2 and STAT5 were determined by Jess Assay analysis. RESULTS: TQ markedly decreased the cell proliferation and induced apoptosis in K562 cells (P < 0.001) in a concentration dependent manner. TQ caused a significant decrease in the transcriptional levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes (P < 0.001). TQ induced a significant decrease in JAK2 and STAT5 protein levels (P < 0.001). CONCLUSION: our results indicated that TQ inhibited cell growth of K562 cells via downregulation of BCR ABL/ JAK2/STAT3 and STAT5 signaling and reducing JAK2 and STAT5 protein levels.

Differential effects of androgens and estrogens over cellular GH sensitivity in HEPG2 cells.

Testosterone and estrogen concentrations progressively increase during puberty, and in association with growth hormone (GH), lead to the increase in height velocity known as the pubertal growth spurt. Very limited information is available however, regarding the possible effects of sex steroids over GH cellular sensitivity. OBJECTIVE: To investigate the effects of different concentrations of testosterone, estradiol and dihydrotestosterone over the GH intracellular signaling pathway. METHODS: We evaluated the effects of these sex steroids on the nuclear phosphorylation of STAT5b and IGF-1 expression, in HEPG2 human hepatoma cells. In addition, we studied whether Tamoxifen (TAM), can modulate these effects. RESULTS: The highest concentration of T tested (10 ng/mL) co-incubated with a fixed concentration of GH (40 ng/mL) increased nuclear STAT5b phosphorylation compared with GH alone (1.34 ± 0.2 vs 0.6 ± 0.09 AU; *p < 0.05), as well as IGF-1 expression (0.6 ± 0.03 vs 0.32 ± 0.05 AU; *p < 0.05). This effect was not observed with lower concentrations of T tested (1 and 5 ng/mL). A similar increase in nuclear STAT5b phosphorylation was observed with the lowest concentration of E2 tested (20 pg/mL), co-incubated with the same fixed concentration of GH (3.6 ± 0.5 vs 1.28 ± 0.33 AU; *p < 0.05). This effect was also associated with an increase in IGF-1 expression (0.73 ± 0.02 vs 0.39 ± 0.04 AU; *p < 0.05). These results were not observed with higher concentrations of E2 tested (75 and 200 pg/mL). DHT at concentrations of 0.1, 0.25 and 0.5 ng/mL, co-stimulated with GH, did not change cytoplasmic STAT5b phosphorylation, nuclear STAT5b or IGF-1 expression. In addition, the co-incubation of TAM with the highest concentration of T tested (10 ng/mL) and GH (40 ng/mL) did not change cytoplasmic, nuclear pSTAT5 levels or IGF-1 expression. CONCLUSIONS: T and E2 potentiate the GH signaling pathway in a concentration-dependent fashion. The observation that the non-aromatizable androgen dihydrotestosterone does not stimulate this pathway, and that the effects of T are blocked with TAM, suggests that the effects of T over the GH signaling pathway appear to be mediated by estrogen.

Suppressing STAT5 signaling affects osteosarcoma growth and stemness.

Osteosarcoma (OS) is the most common primary bone tumor that primarily affects children and adolescents. Studies suggested that dysregulation JAK/STAT signaling promotes the development of OS. Cells treated with pimozide, a STAT5 inhibitor suppressed proliferation and colony formation and induced sub G0/G1 cell cycle arrest and apoptosis. There was a reduction in cyclin D1 and CDK2 expression and Rb phosphorylation, and activation of Caspase-3 and PARP cleavage. In addition, pimozide suppressed the formation of 3-dimensional osteospheres and growth of the cells in the Tumor in a Dish lung organoid system. Furthermore, there was a reduction in expression of cancer stem cell marker proteins DCLK1, CD44, CD133, Oct-4, and ABCG2. More importantly, it was the short form of DCLK1 that was upregulated in osteospheres, which was suppressed in response to pimozide. We further confirmed by flow cytometry a reduction in DCLK1+ cells. Moreover, pimozide inhibits the phosphorylation of STAT5, STAT3, and ERK in OS cells. Molecular docking studies suggest that pimozide interacts with STAT5A and STAT5B with binding energies of -8.4 and -6.4 Kcal/mol, respectively. Binding was confirmed by cellular thermal shift assay. To further understand the role of STAT5, we knocked down the two isoforms using specific siRNAs. While knockdown of the proteins did not affect the cells, knockdown of STAT5B reduced pimozide-induced necrosis and further enhanced late apoptosis. To determine the effect of pimozide on tumor growth in vivo, we administered pimozide intraperitoneally at a dose of 10 mg/kg BW every day for 21 days in mice carrying KHOS/NP tumor xenografts. Pimozide treatment significantly suppressed xenograft growth. Western blot and immunohistochemistry analyses also demonstrated significant inhibition of stem cell marker proteins. Together, these data suggest that pimozide treatment suppresses OS growth by targeting both proliferating cells and stem cells at least in part by inhibiting the STAT5 signaling pathway.

Growth hormone activates X-box binding protein 1 in a sexually dimorphic manner through the extracellular signal-regulated protein kinase and CCAAT/enhancer-binding protein ß pathway in rat liver.

Growth hormone (GH) has multiple physiological roles, acting on many organs. In order to investigate its roles in rat liver, we tried to identify novel genes whose transcription was regulated by GH. We identified X-box binding protein 1 (Xbp1) as a candidate gene. XBP1 is a key transcription factor activated in response to endoplasmic reticulum (ER) stress. The purpose of this study was to investigate the mode of action of GH on XBP1, including the relation with ER stress, sex-dependent expression of the mRNA, and the signaling pathway. Intravenous administration of GH rapidly and transiently increased Xbp1 mRNA in hypophysectomized rat livers. Neither phosphorylated inositol-requiring-1α (IRE1α) nor phosphorylated PKR-like ER kinase (PERK) increased, suggesting that Xbp1 expression is induced by an ER stress-independent mechanism. The active form of XBP1(S) protein was increased by GH administration and was followed by an increased ER-associated dnaJ protein 4 (ERdj4) mRNA level. XBP1(S) protein levels were predominantly identified in male rat livers with variations among individuals similar to those of phosphorylated signal transducer and activator of transcription 5B (STAT5B), suggesting that XBP1(S) protein levels are regulated by the sex-dependent secretary pattern of GH. The GH signaling pathway to induce Xbp1 mRNA was examined in rat hepatoma H4IIE cells. GH induced the phosphorylation of CCAAT/enhancer-binding protein ß (C/EBPß) following extracellular signal-regulated protein kinase (ERK) phosphorylation. Taken together, the results indicated that XBP1 is activated by GH in rat liver in a sexually dimorphic manner via ERK and C/EBPß pathway.

IL-7-Mediated IL-7R-JAK3/STAT5 signalling pathway contributes to chemotherapeutic sensitivity in non-small-cell lung cancer.

OBJECTIVES: The chemotherapy drug resistance is a major challenge for non-small-cell lung cancer (NSCLC) treatment. Combination of immunotherapy and chemotherapy has shown promise for cancer. The goal of this study was to evaluate the anti-tumour efficacy of interleukin-7 (IL-7) combining cisplatin against NSCLC. MATERIALS AND METHODS: Cell proliferation was analysed using CCK-8 assay, EdU proliferation assay and colony-forming assay. Cell apoptosis was evaluated using HOECHST 33342 assay and flow cytometry. The protein expression levels were analysed by Western blot. The blocking antibody against the IL-7 receptor and the inhibitors of STAT5 and JAK3 were used to investigate the pathway involved. A xenograft model was established to assess the anti-tumour efficacy of IL-7 combining cisplatin in vivo. RESULTS: Here we found IL-7R was increased in A549/DDP cells compared with A549 cells. The block of IL-7R reversed the inhibitory effects of IL-7 combined with cisplatin and decreased the numbers of apoptosis cells induced by treatment of IL-7 combined with cisplatin. The JAK3 inhibitor and STAT5 inhibitor were used to identify the pathway involved. The results showed that JAK3/STAT5 pathway was involved in enhancing role of cisplatin sensitivity of NSCLC cells by IL-7. In vivo, cisplatin significantly inhibited tumour growth and IL-7 combined with cisplatin achieved the best therapeutic effect. CONCLUSION: Together, IL-7 promoted the sensitivity of NSCLC cells to cisplatin via IL-7R-JAK3/STAT5 signalling pathway.

Bisphenol S Alters the Lactating Mammary Gland and Nursing Behaviors in Mice Exposed During Pregnancy and Lactation.

High doses of estrogenic pharmaceuticals were once prescribed to women to halt lactation. Yet, the effects of low-level xenoestrogens on lactation remain poorly studied. We investigated the effects of bisphenol S (BPS), an estrogen receptor (ER) agonist, on the lactating mammary gland; the arcuate nucleus, a region of the hypothalamus important for neuroendocrine control of lactational behaviors; and nursing behavior in CD-1 mice. Female mice were exposed to vehicle, 2 or 200 µg BPS/kg/d from pregnancy day 9 until lactational day (LD) 20, and tissues were collected on LD21. Tissues were also collected from a second group at LD2. BPS exposure significantly reduced the fraction of the mammary gland comprised of lobules, the milk-producing units, on LD21, but not LD2. BPS also altered expression of Esr1 and ERα in the mammary gland at LD21, consistent with early involution. In the arcuate nucleus, no changes were observed in expression of signal transducer and activator of transcription 5, a marker of prolactin signaling, or ERα, suggesting that BPS may act directly on the mammary gland. However, observations of nursing behavior collected during the lactational period revealed stage-specific effects on both pup and maternal nursing behaviors; BPS-treated dams spent significantly more time nursing later in the lactational period, and BPS-treated pups were less likely to initiate nursing. Pup growth and development were also stunted. These data indicate that low doses of BPS can alter lactational behaviors and the maternal mammary gland. Together, they support the hypothesis that pregnancy and lactation are sensitive to low-dose xenoestrogen exposures.

Resveratrol attenuates constitutive STAT3 and STAT5 activation through induction of PTPε and SHP-2 tyrosine phosphatases and potentiates sorafenib-induced apoptosis in renal cell carcinoma.

BACKGROUND: Signal transducers and activators of transcription (STAT) proteins are critical transcription factor that are aberrantly activated in various types of malignancies, including renal cell carcinoma (RCC). METHODS: We investigated the effect of resveratrol (RES), an edible polyphenol phytoalexin on STAT3 and STAT5 activation cascade in both Caki-1 and 786-O RCC cell lines. RESULTS: We found that RES suppressed both constitutive STAT3 (tyrosine residue 705 and serine residue 727) and STAT5 (tyrosine residue 694 and 699) activation, which correlated with the suppression of the upstream kinases (JAK1, JAK2, and c-Src) in RCC. Also, RES abrogated DNA binding capacity and nuclear translocation of these two transcription factors. RES-induced an increased expression of PTPε and SHP-2 and the deletion of these two genes by small interfering RNA abolished the ability of RES to inhibit STAT3 activation, suggesting the critical role of both PTPε and SHP-2 in its possible mechanism of action. Moreover, RES induced S phase cell cycle arrest, caused induction of apoptosis, loss of mitochondrial membrane potential, and suppressed colony formation in RCC. We also found that RES downregulated the expression of STAT3/5-regulated antiapoptotic, proliferative, and metastatic gene products; and this correlated with induction of caspase-3 activation and anti-invasive activity. Beside, RES potentiated sorafenib induced inhibitory effect on constitutive STAT3 and STAT5 phosphorylation, apoptotic effects in 786-O cells, and this correlated with down-regulation of various oncogenic gene products. CONCLUSION: Overall, our results suggest that RES is a blocker of both STAT3 and STAT5 activation and thus may exert potential growth inhibitory effects against RCC cells.

GH signaling in human adipose and muscle tissue during 'feast and famine': amplification of exercise stimulation following fasting compared to glucose administration.

OBJECTIVE: Fasting and exercise stimulates, whereas glucose suppresses GH secretion, but it is uncertain how these conditions impact GH signaling in peripheral tissues. To test the original 'feast and famine hypothesis' by Rabinowitz and Zierler, according to which the metabolic effects of GH are predominant during fasting, we specifically hypothesized that fasting and exercise act in synergy to increase STAT-5b target gene expression. DESIGN AND METHODS: Eight healthy men were studied on two occasions in relation to a 1 h exercise bout: i) with a concomitant i.v. glucose infusion ('feast') and ii) after a 36 h fast ('famine'). Muscle and fat biopsy specimens were obtained before, immediately after, and 30 min after exercise. RESULTS: GH increased during exercise on both examination days and this effect was amplified by fasting, and free fatty acid (FFA) levels increased after fasting. STAT-5b phosphorylation increased similarly following exercise on both occasions. In adipose tissue, suppressors of cytokine signaling 1 (SOCS1) and SOCS2 were increased after exercise on the fasting day and both fasting and exercise increased cytokine inducible SH2-containing protein (CISH). In muscle, SOCS2 and CISH mRNA were persistently increased after fasting. Muscle SOCS1, SOCS3, and CISH mRNA expression increased, whereas SOCS2 decreased after exercise on both examination days. CONCLUSIONS: This study demonstrates that fasting and exercise act in tandem to amplify STAT-5b target gene expression (SOCS and CISH) in adipose and muscle tissue in accordance with the 'feast and famine hypothesis'; the adipose tissue signaling responses, which hitherto have not been scrutinized, may play a particular role in promoting FFA mobilization.

The MEK1/2 inhibitor U0126 reverses imatinib resistance through down-regulating activation of Lyn/ERK signaling pathway in imatinib-resistant K562R leukemia cells.

Chronic myelogenous leukemia (CML) is triggered by the constitutively activated BCR-ABL oncoprotein and multiple downstream signaling pathways, including the Raf/MEK/ERK, Akt/mTOR, SRC, and STAT5 pathways. The BCR-ABL tyrosine kinase inhibitor imatinib is the standard treatment for CML. However, the development of imatinib resistance has become a new challenge for CML treatment. Here, we investigated the expression levels of the signaling pathways to explore the cause of imatinib resistance and seek new reversing drugs. Our results showed that abnormal activation of the BCR-ABL-independent Lyn/ERK signaling pathway was involved in imatinib-resistance of K562R cells. Furthermore, p-Lyn and p-ERK were up-regulated after treatment with imatinib alone. However, U0126, a MEK1/2 inhibitor, could counteract the up-regulation induced by imatinib, and the combination of imatinib and U0126 could overcome the resistance to imatinib in K562R cells. In conclusion, our studies suggest that the combination of imatinib and an inhibitor of the ERK signaling pathway may be effective in imatinib-resistant CML patients.

JAK1/2 and Pan-deacetylase inhibitor combination therapy yields improved efficacy in preclinical mouse models of JAK2V617F-driven disease.

PURPOSE: The myeloproliferative neoplasm myelofibrosis is characterized by frequent deregulation of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, and JAK inhibitors were shown to reduce splenomegaly and ameliorate disease-related symptoms. However, the mutant clone and bone marrow fibrosis persist in the majority of patients. Using preclinical models, we explored whether JAK and pan-deacetylase inhibitor combination yielded additional benefits. EXPERIMENTAL DESIGN: The combination of the JAK1/2 inhibitor ruxolitinib and panobinostat was investigated using two different mouse models of JAK2(V617F)-driven disease. A Ba/F3 JAK2(V617F) cell-driven leukemic disease model was used to identify tolerated and efficacious doses. The drugs were then evaluated alone and in combination in a mouse model of myeloproliferative neoplasm-like disease based on transplantation of bone marrow transduced with a retrovirus expressing JAK2(V617F). Exposures were determined in blood and tissues, and phosphorylated STAT5 and acetylated histone H3 pharmacodynamic readouts were assessed in spleen and bone marrow. Histologic analysis was conducted on spleen and bone marrow, including staining of reticulin fibers in the latter organ. RESULTS: The combination of ruxolitinib and panobinostat was found to have a more profound effect on splenomegaly, as well as on bone marrow and spleen histology, compared with either agent alone, and the analysis of pharmacodynamic readouts showed that ruxolitinib and panobinostat have nonoverlapping and complementary effects. CONCLUSION: Combining JAK1/2 and pan-deacetylase inhibitors was fairly well tolerated and resulted in improved efficacy in mouse models of JAK2(V617F)-driven disease compared with the single agents. Thus, the combination of ruxolitinib and panobinostat may represent a promising novel therapeutic modality for myeloproliferative neoplasms.

Differentiation therapy: targeting human renal cancer stem cells with interleukin 15.

BACKGROUND: Many renal cancer patients experience disease recurrence after immunotherapy or combined treatments due to persistence of cancer stem cells (CSCs). The identification of reliable inducers of CSC differentiation may facilitate the development of efficient strategies for eliminating CSCs. We investigated whether interleukin 15 (IL-15), a regulator of kidney homeostasis, induces the differentiation of CD105-positive (CD105(+)) CSCs from human renal cancers. METHODS: CD105(+) CSCs were cultured to preserve their stem cell properties and treated with recombinant human IL-15 (rhIL-15) to evaluate their ability to differentiate, to acquire sensitivity to chemotherapeutic drugs, and to form spheroids in vitro and tumors in vivo. Expression of stem cell and epithelial markers were studied by flow cytometry, immunocytochemistry, and immunoblotting. Identification of a CSC side population fraction and its sensitivity to chemotherapy drugs and expression of ATP-binding cassette (ABC) transporters and aldehyde dehydrogenase (ALDH) activities were determined by flow cytometry. Spheroid formation was determined in limiting dilution assay. Xenograft tumors were generated in severe combined immunodeficient mice (n = 12-18 mice per group). All statistical tests were two-sided. RESULTS: CD105(+) CSCs treated with rhIL-15 at 10 pg/mL differentiated into cells expressing epithelial markers. rhIL-15 induced epithelial differentiation of all CD105(+) CSCs subsets and blocked CSC self-renewal (sphere-forming ability) and their tumorigenic properties in severe combined immunodeficient mice. Vinblastine and paclitaxel induced statistically significant higher levels of apoptosis in rhIL-15-differentiated epithelial cells compared with CD105(+) CSCs (mean percentage of apoptotic cells, vinblastine: 33% vs 16.5%, difference = 16.5%, 95% confidence interval = 12.25% to 20.74%, P = .0025; paclitaxel: 35% vs 11.6%, difference = 23.4%, 95% confidence interval = 22.5% to 24.24%, P = .0015). The higher sensitivity of rhIL-15-differentiated epithelial cells to chemotherapeutic drugs was associated with loss of detoxifying mechanisms such as ALDH and ABC transporter activities. CONCLUSION: IL-15 directs the epithelial differentiation of renal CSCs and meets the criteria for a treatment strategy: CSC pool depletion and generation of differentiated nontumorigenic cells that are sensitive to chemotherapeutic agents.

Effective targeting of STAT5-mediated survival in myeloproliferative neoplasms using ABT-737 combined with rapamycin.

Signal transducer and activator of transcription-5 (STAT5) is a critical transcription factor for normal hematopoiesis and its sustained activation is associated with hematologic malignancy. A persistently active mutant of STAT5 (STAT5a(S711F)) associates with Grb2-associated binding protein 2 (Gab2) in myeloid leukemias and promotes growth in vitro through AKT activation. Here we have retrovirally transduced wild-type or Gab2(-/-) mouse bone marrow cells expressing STAT5a(S711F) and transplanted into irradiated recipient mice to test an in vivo myeloproliferative disease model. To target Gab2-independent AKT/mTOR activation, we treated wild-type mice separately with rapamycin. In either case, mice lacking Gab2 or treated with rapamycin showed attenuated myeloid hyperplasia and modestly improved survival, but the effects were not cytotoxic and were reversible. To improve on this approach, we combined in vitro targeting of STAT5-mediated AKT/mTOR using rapamycin with inhibition of the STAT5 direct target genes bcl-2 and bcl-X(L) using ABT-737. Striking synergy with both drugs was observed in mouse BaF3 cells expressing STAT5a(S711F), TEL-JAK2 or BCR-ABL and in the relatively single agent-resistant human BCR-ABL-positive K562 cell line. Therefore, targeting distinct STAT5-mediated survival signals, for example, bcl-2/bcl-X(L) and AKT/mTOR may be an effective therapeutic approach for human myeloproliferative neoplasms.

Erythropoietin-induced activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK pathways promotes malignant cell behavior in a modified breast cancer cell line.

Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

Celecoxib activates Stat5 and restores or increases the expression of growth hormone-regulated genes in hepatocarcinogenesis.

We have previously evaluated the chemopreventive effect of celecoxib on preneoplastic lesions in rat liver. However, though the effects of celecoxib have been tested in a variety of carcinomas, there has not been a study on the modulation of gene expression in response to this drug. Here, we evaluated the effect of celecoxib on the gene expression profile associated with hepatocarcinogenesis. Male Sprague-Dawley rats underwent the modified resistant hepatocyte model and were fed a diet containing 1500 ppm of celecoxib. Gene expression profiles were evaluated using DNA microarrays and further validations were performed using quantitative PCR, western blotting and immunohistochemical staining. Celecoxib modulated the expression of 46 genes, and those regulated by growth hormone were selected for further analysis. Celecoxib significantly upregulated the expression of the Cyp2b1/2, Cyp3a1, and alpha2-urinary globulin (alpha2uG) genes and restored the expression of Cyp2b3 to normal. The protein expression of Cyp2b1/2 was increased, but the expressions of Cyp3a1 and alpha2uG were only restored to normal levels. The increased Cyp2b1/2 expression in response to celecoxib was mainly confined to preneoplastic lesions. A search for the upstream mediator of these genetic alterations found that carcinogenesis inactivated by 87% the signal transducer and activator of transcription 5 (Stat5), a transcription factor that is activated by growth hormone signaling, but celecoxib treatment restored its activation. In conclusion, these results suggest that celecoxib exerts anticancer effects on altered hepatic cells by restoring mRNA and the protein expression levels of specific genes, in part through the reactivation of Stat5.

Development of a novel ligand that activates JAK2/STAT5 signaling through a heterodimer of prolactin receptor and growth hormone receptor.

The objective of this study was to determine if a functional heterodimer of prolactin receptor (PRLR) and growth hormone receptor (GHR) can be formed in humans. A novel ligand was designed that is composed of a GHR antagonist (B2036) and a PRLR antagonist (G129R) fused in tandem (B2036-G129R). Because both B2036 and G129R are binding site 2 inactive antagonists, the B2036-G129R fusion protein, in theory contains only two functional binding site 1s: one for GHR and one for PRLR. We examined the behavior of this chimeric ligand in cell lines known to express GHR, PRLR, or both receptors. The data presented show that B2036-G129R is inactive in IM-9 cells that express only GHR or Nb2 cells that express PRLR. In T-47D cells that coexpress PRLR and GHR, B2036-G129R activates JAK2/STAT5 signaling. These findings provide evidence that B2036-G129R is able to activate signal transduction through a heterodimer of PRLR and GHR in humans.

Imatinib mesylate inhibits CD4+ CD25+ regulatory T cell activity and enhances active immunotherapy against BCR-ABL- tumors.

Imatinib mesylate (Gleevec, STI571), a selective inhibitor of a restricted number of tyrosine kinases, has been effectively used for the treatment of Philadelphia chromosome-positive leukemias and gastrointestinal stromal tumors. Imatinib may also directly influence immune cells. Suppressive as well as stimulating effects of this drug on CD4(+) and CD8(+) T lymphocytes or dendritic cells have been reported. In the current study, we have investigated the influence of imatinib mesylate on CD4(+)CD25(+)FoxP3(+) regulatory T cells (Treg), a critical population of lymphocytes that contributes to peripheral tolerance. Used at concentrations achieved clinically, imatinib impaired Treg immunosuppressive function and FoxP3 expression but not production of IL-10 and TGF-beta in vitro. Imatinib significantly reduced the activation of the transcription factors STAT3 and STAT5 in Treg. Analysis of Treg TCR-induced signaling cascade indicated that imatinib inhibited phosphorylation of ZAP70 and LAT. Substantiating these observations, imatinib treatment of mice decreased Treg frequency and impaired their immunosuppressive function in vivo. Furthermore, imatinib mesylate significantly enhanced antitumor immune responses to dendritic cell-based immunization against an imatinib-resistant BCR-ABL negative lymphoma. The clinical applications of imatinib mesylate might thus be expanded with its use as a potent immunomodulatory agent targeting Treg in cancer immunotherapy.

Enhancement of hypoxia-induced apoptosis of human breast cancer cells via STAT5b by momilactone B.

We have shown previously that hypoxia activates the cyclin D1 promoter via the Jak2/STAT5b pathway in breast cancer cells. Most solid tumors contain hypoxic components and overexpression of cyclin D1. The purpose of the present study was to investigate the molecular mechanism by which momilactone B exerts its inhibitory effects on breast cancer cells. Momilactone B, extracted from Korean rice hulls, suppressed hypoxia-induced increases in phospho-STAT5, STAT5b, cyclin D1, and cdk4 protein levels in human breast cancer cells. STAT5b expression was inhibited by siRNA experiments leading to decreased cyclin D1. The effects of momilactone B on cell growth and apoptosis-related gene expression were investigated in breast cancer cells under hypoxic conditions (2% O2). Bax and p21 expression was found to be up-regulated, whereas ppRb and bcl-2 were down-regulated in momilactone B-treated cells under hypoxic conditions. However, the p53 protein level did not change. Flow cytometry with Annexin-FITC staining showed that the number of apoptotic cells increased in hypoxic cells treated with momilactone B compared with untreated hypoxic cells. Furthermore, caspase activity increased upon treatment with momilactone B under hypoxic conditions. These results indicate that momilactone B inhibits the growth of breast cancer cells, regulates the expression of apoptosis-related genes, and induces apoptosis through STAT5b and a caspase-3 dependent pathway. We suggest that momilactone B accelerates hypoxia-induced apoptosis of human breast cancer cells through STAT5b, and may represent an effective chemopreventive or therapeutic agent against breast cancer.

The multikinase inhibitor sorafenib induces apoptosis in highly imatinib mesylate-resistant bcr/abl+ human leukemia cells in association with signal transducer and activator of transcription 5 inhibition and myeloid cell leukemia-1 down-regulation.

The effects of the multikinase inhibitor sorafenib (BAY 43-9006), an agent shown previously to induce apoptosis in human leukemia cells through inhibition of myeloid cell leukemia-1 (Mcl-1) translation, have been examined in Bcr/Abl(+) leukemia cells resistant to imatinib mesylate (IM). When administered at pharmacologically relevant concentrations (10-15 microM), sorafenib potently induced apoptosis in imatinib mesylate-resistant cells expressing high levels of Bcr/Abl, cells exhibiting a Bcr/Abl-independent, Lyn-dependent form of resistance, and CD34(+) cells obtained from imatinib-resistant patients. In addition, Ba/F3 cells expressing mutations rendering them resistant to IM (e.g., E255K, M351T) or to IM, dasatinib, and nilotinib (T315I) remained fully sensitive to sorafenib. Induction of apoptosis by sorafenib was associated with rapid and pronounced down-regulation of Mcl-1 and diminished signal transducer and activator of transcription (STAT) 5 phosphorylation and reporter activity but only very modest and delayed inactivation of the Bcr/Abl downstream target Crkl. Moreover, transfection with a constitutively active STAT5 construct partially but significantly protected cells from sorafenib lethality. Ba/F3 cells expressing Bcr/Abl mutations were as sensitive to sorafenib-induced Mcl-1 down-regulation and dephosphorylation of STAT5 and eukaryotic initiation factor 4E as wild-type cells. Finally, stable knockdown of Bcl-2-interacting mediator of cell death (Bim) with short hairpin RNA in K562 cells significantly diminished sorafenib lethality, arguing strongly for a functional role of this proapoptotic Bcl-2 family member in the lethality of this agent. Together, these findings suggest that sorafenib effectively induces apoptosis in highly imatinib-resistant chronic myelogenous leukemia cells, most likely by inhibiting or down-regulating targets (i.e., STAT5 and Mcl-1) downstream or independent of Bcr/Abl.

Functional and immunochemical characterisation of different antibodies against the erythropoietin receptor.

Since it was discovered that the hematopoietic hormone erythropoetin (EPO) exerts neuroprotective effects in the CNS, many studies on the EPO receptor (EPOR) function and localisation in the CNS have been performed. For this purpose, commercially available anti-EPOR antibodies have often been applied. As the literature data on these antibodies show inconsistencies, we here systematically compared six frequently used, commercially available EPOR antibodies for different applications. Five of the antibodies appeared to specifically recognize recombinant rat and human EPOR in HEK293 cells by Western blotting, but the same antibodies yielded different and inconsistent results when human UT-7 cells or rat brain tissue were applied. Immunocytochemical staining of EPOR-transfected HEK cells only produced consistent results with three of the six antibodies. All antibodies stained neurons in rat brain sections, but with large differences in the staining pattern and only the C-20 EPOR antibody was found to label astrocytes. Since EPOR antibodies have been applied in several studies as EPOR antagonists, we further tested the antibodies for their capacity to functionally block the EPO-EPOR interaction in a cellular signalling system with STAT-5 phosphorylation as readout. Here, only the MAB307 antibody showed a partial effect at concentrations of 5-50 microg/ml.