Curcumol Attenuates Endometriosis by Inhibiting the JAK2/STAT3 Signaling Pathway.

BACKGROUND Curcumol is a hydrogenated austenitic compound with hemiketal. In this study we evaluated the effects of curcumol on local inflammatory response, cell proliferation, and metastasis in endometriosis, and elucidated the underlying mechanisms. MATERIAL AND METHODS Ectopic endometrial stromal cells were treated with increasing doses of curcumol. The MTT assay was used to assess cell viability. FITC-labeled annexin-V/PI double-staining method and flow cytometry were used to determine cell apoptosis. Cell migration was evaluated using a wound healing assay. ELISA kits were used to detect the levels of TNF-alpha, IL-6, and IL-1ß. Western blot assay was used to examine the phosphorylation degree of JAK2 and STAT3 and the expression of Bax, Bcl2, and caspase-3 proteins. Autologous endometrial transplantation was used to establish a rat model to assess the anti-EMS effect of curcumol in vivo. RESULTS Curcumol can inhibit the proliferation of ectopic endometrial stromal cells, promote cell apoptosis, and weaken cell migration ability. Curcumol can reduce the expression of Bax and caspase-3 protein and increase the expression of Bcl2 protein. Curcumol also can inhibit the secretion of inflammatory cytokines, including tumor necrosis cytokines (TNF)-alpha, interleukin (IL)-6, and IL-1ß, by ectopic endometrial stromal cells. In addition, curcumol can also inhibit the phosphorylation of JAK2 and STAT3. In vivo experiments also proved that curcumol could inhibit the growth of ectopic lesions in EMS model rats. CONCLUSIONS Curcumol can inhibit the JAK2/STAT3 pathway, reduce the inflammatory cytokines secreted by ectopic endometrial stromal cells, inhibit cell proliferation and migration, and reduce the volume of ectopic lesions.

Red nucleus IL-33 facilitates the early development of mononeuropathic pain in male rats by inducing TNF-α through activating ERK, p38 MAPK, and JAK2/STAT3.

BACKGROUND: Our recent studies have identified that the red nucleus (RN) dual-directionally modulates the development and maintenance of mononeuropathic pain through secreting proinflammatory and anti-inflammatory cytokines. Here, we further explored the action of red nucleus IL-33 in the early development of mononeuropathic pain. METHODS: In this study, male rats with spared nerve injury (SNI) were used as mononeuropathic pain model. Immunohistochemistry, Western blotting, and behavioral testing were used to assess the expressions, cellular distributions, and actions of red nucleus IL-33 and its related downstream signaling molecules. RESULTS: IL-33 and its receptor ST2 were constitutively expressed in the RN in naive rats. After SNI, both IL-33 and ST2 were upregulated significantly at 3 days and peaked at 1 week post-injury, especially in RN neurons, oligodendrocytes, and microglia. Blockade of red nucleus IL-33 with anti-IL-33 neutralizing antibody attenuated SNI-induced mononeuropathic pain, while intrarubral administration of exogenous IL-33 evoked mechanical hypersensitivity in naive rats. Red nucleus IL-33 generated an algesic effect in the early development of SNI-induced mononeuropathic pain through activating NF-κB, ERK, p38 MAPK, and JAK2/STAT3, suppression of NF-κB, ERK, p38 MAPK, and JAK2/STAT3 with corresponding inhibitors markedly attenuated SNI-induced mononeuropathic pain or IL-33-evoked mechanical hypersensitivity in naive rats. Red nucleus IL-33 contributed to SNI-induced mononeuropathic pain by stimulating TNF-α expression, which could be abolished by administration of inhibitors against ERK, p38 MAPK, and JAK2/STAT3, but not NF-κB. CONCLUSIONS: These results suggest that red nucleus IL-33 facilitates the early development of mononeuropathic pain through activating NF-κB, ERK, p38 MAPK, and JAK2/STAT3. IL-33 mediates algesic effect partly by inducing TNF-α through activating ERK, p38 MAPK and JAK2/STAT3.

Cutaneous JAK Expression in Vitiligo.

BACKGROUND: The Janus kinase-signal transducer and activator of transcription signaling pathway has been suggested as a promising therapeutic target in vitiligo. However, limited data is available on the cutaneous expression of JAK in vitiligo. AIM: This study is designed to analyze the cutaneous expression patterns of JAK1, 2, and 3 in vitiligo and investigate their relation to the disease clinical parameters. METHODS: This case-control study recruited 24 patients having active vitiligo and 20 age, sex, and skin type-matched healthy volunteers. Skin biopsies were obtained from patients (lesional, perilesional and nonlesional) and controls for assessment of JAK1, 2, and 3 expression using RT-PCR. RESULTS: JAK1 and JAK3 were overexpressed in patients' skin compared to control skin and showed a stepwise pattern of upregulation from control to nonlesional, perilesional and lesional skin. However, JAK3 showed much stronger expression. In contrast JAK2 expression showed no significant difference in any of lesional, perilesional or nonlesional skin compared to control skin. JAK1 and JAK3 expression levels showed no correlation with neither the disease activity nor severity. CONCLUSION: JAK1 and more prominently JAK3 are upregulated in vitiliginous skin and possibly contribute to the pathogenesis of the disease. Accordingly, selective JAK3/1 inhibition may provide a favorable therapeutic opportunity for vitiligo patients.This study is registered on the ClinicalTrials.gov Identifier: NCT03185312.

Revisiting bupropion anti-inflammatory action: involvement of the TLR2/TLR4 and JAK2/STAT3.

There are accumulating reports regarding poor response to common antidepressant therapy. Antidepressant resistance is often linked to inflammatory system activation and patients displaying inflammation prior to the treatment are less responsive to antidepressants. We hypothesized that the inefficacy of antidepressant therapy in some patients may be attributable to the drugs' inflammatory mode of action, which has been overlooked because of their substantial therapeutic benefit. Bupropion is a commonly prescribed antidepressant that is often used to treat seasonal affective disorders as well. Nevertheless, research suggests that bupropion causes inflammation and worsens depressive symptoms. Therefore, we investigated the impact of bupropion on cytokines of innate and adaptive immunity, as well as immune signaling pathways. We treated lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs) with different doses of bupropion. Pro-/anti-inflammatory cytokines [tumor necrosis factor alpha (TNFα), interleukin-1ß (IL-1ß), IL-17, and IL-10] were assessed at both transcriptional and translational levels as well as the involvement of JAK2 /STAT3, TLR2, and TLR4 signaling in this process. Bupropion reduced IL-17A, TNFα, and IL-1ß protein levels in the cultures. Nonetheless, bupropion increased IL-1ß (P < 0.0001), TNFα (P < 0.0001), and IL-17A (P < 0.05) mRNA levels. Treatment enhanced both IL-10 concentration (P < 0.0001) and gene expression (P < 0.0001). TLR2 (P < 0.0001), TLR4 (P < 0.0001), JAK2 (P < 0.0001), and STAT3 (P < 0.0001) gene expression also rose in response to bupropion. The findings imply that bupropion, particularly 50 µM and 100 µM, has pro-inflammatory effects and should be co-administered with anti-inflammatory medications, at least in patients with inflammatory conditions.

Aminocyanopyridines as anti-lung cancer agents by inhibiting the STAT3 pathway.

Lung cancer is a leading cause of cancer-related death worldwide. Cyanopyridines and aminocyanopyridines with carbon-nitrogen bonds have been proved to exert significant anticancer, antibacterial, and anti-inflammatory effects. In this study, we showed that aminocyanopyridine 3o and 3k displaying potent antitumor activity via inhibiting the signal transducer and activator of transcription 3 (STAT3) pathway. They blocked the constitutive STAT3 phosphorylation in a dose- and time-dependent manner and regulated the transcription of STAT3 target genes encoding apoptosis factors. Most importantly, 3o also inhibited interleukin-6-induced STAT3 activation and nuclear localization. Furthermore, 3o significantly inhibited the tumor growth of H460-derived xenografts. Taken together, these findings suggest that 3o and 3k are promising therapeutic drug candidates for lung cancer by inhibiting persistent STAT3 signaling.

Vascular endothelial cell expression of JAK2V617F is sufficient to promote a pro-thrombotic state due to increased P-selectin expression.

Thrombosis is the main cause of morbidity and mortality in patients with JAK2V617F myeloproliferative neoplasms. Recent studies have reported the presence of JAK2V617F in endothelial cells of some patients with myeloproliferative neoplasms. We investigated the role of endothelial cells that express JAK2V617F in thrombus formation using an in vitro model of human endothelial cells overexpressing JAK2V617F and an in vivo model of mice with endothelial-specific JAK2V617F expression. Interestingly, these mice displayed a higher propensity for thrombus. When deciphering the mechanisms by which JAK2V617F-expressing endothelial cells promote thrombosis, we observed that they have a pro-adhesive phenotype associated with increased endothelial P-selectin exposure, secondary to degranulation of Weibel-Palade bodies. We demonstrated that P-selectin blockade was sufficient to reduce the increased propensity of thrombosis. Moreover, treatment with hydroxyurea also reduced thrombosis and decreased the pathological interaction between leukocytes and JAK2V617F-expressing endothelial cells through direct reduction of endothelial P-selectin expression. Taken together, our data provide evidence that JAK2V617F-expressing endothelial cells promote thrombosis through induction of endothelial P-selectin expression, which can be reversed by hydroxyurea. Our findings increase our understanding of thrombosis in patients with myeloproliferative neoplasms, at least those with JAK2V617F-positive endothelial cells, and highlight a new role for hydroxyurea. This novel finding provides the proof of concept that an acquired genetic mutation can affect the pro-thrombotic nature of endothelial cells, suggesting that other mutations in endothelial cells could be causal in thrombotic disorders of unknown cause, which account for 50% of recurrent venous thromboses.

Colony-Forming Cell Assay Detecting the Co-Expression of JAK2V617F and BCR-ABL1 in the Same Clone: A Case Report.

BCR-ABL1-negative myeloproliferative disorders and chronic myeloid leukaemia are haematologic malignancies characterised by single and mutually exclusive genetic alterations. Nevertheless, several patients co-expressing the JAK2V617F mutation and the BCR-ABL1 transcript have been described in the literature. We report the case of a 61-year-old male who presented with an essential thrombocythaemia phenotype and had a subsequent diagnosis of chronic phase chronic myeloid leukaemia. Colony-forming assays demonstrated the coexistence of 2 different haematopoietic clones: one was positive for the JAK2V617F mutation and the other co-expressed both JAK2V617F and the BCR-ABL1 fusion gene. No colonies displayed the BCR-ABL1 transcript alone. These findings indicate that the JAK2V617F mutation was the founding genetic alteration of the disease, followed by the acquisition of the BCR-ABL1 chimeric oncogene. Our data support the hypothesis that a heterozygous JAK2V617F clone may have favoured the bi-clonal nature of this myeloproliferative disorder, generating clones harbouring a second transforming genetic event.

CD274 (PDL1) and JAK2 genomic amplifications in pulmonary squamous-cell and adenocarcinoma patients.

AIMS: CD274 (PDL1) and JAK2 (9p24.1) gene amplifications have been recently described in pulmonary carcinomas in association with programmed death-ligand 1 (PD-L1) expression. Furthermore, PTEN loss has been explored preclinically in relation to PD-L1 expression. Our aim was to determine whether these genomic alterations affect PD-L1 expression levels in non-small-cell lung cancer. METHODS AND RESULTS: PD-L1 and PTEN expression determined by immunohistochemistry (IHC), and CD274, JAK2 and PTEN copy number alterations (CNAs) determined by fluorescence in-situ hybridisation, were studied in 171 pulmonary carcinoma specimens. PD-L1 expression was positive in 40 cases (23.3%), and CD274 amplification was present in 14 tumours (8.8%). Concordance between both events was found in 12 of 14 amplified cases (P = 0.0001). We found nine JAK2-amplified cases (5.7%), seven with PD-L1 expression (P = 0.0006). Moreover, six of the seven cases had JAK2 and CD274 coamplification (9p24.1 genomic amplification). Remarkably, the average PD-L1 IHC score was higher in these amplified cases (230 versus 80; P = 0.001). Non-statistical associations were observed between PD-L1 expression and PTEN loss and PTEN deletions. CONCLUSIONS: We describe a subset of patients (8.2%) who had 9p24.1 amplifications resulting in high expression of PD-L1. Our results provide evidence for genomic up-regulation of PD-L1 expression in non-small-cell lung cancer.

JAK2 variations and functions in lung adenocarcinoma.

Lung cancer ranks as the first most common cancer and the first leading cause of cancer-related death in China and worldwide. Due to the difficulty in early diagnosis and the onset of cancer metastasis, the 5-year survival rate of lung cancer remains low. JAK2 has emerged as pivotal participant in biological processes, often dysregulated in a range of cancers. Recently our study found that JAK2 might play an important role in lung cancer pathogenesis. While our understanding of JAK2 in the onset and progression of lung cancer is still in its infancy, there is no doubt that understanding the variations and functions of JAK2 will certainly secure strong biomarkers and improve treatment options for lung cancer patients. The expression level of JAK2 mRNA was assayed using RT-PCR. JAK2 mutations and amplification were detected using next-generation sequencing (NGS). The shRNA and overexpression plasmids of JAK2 were conducted. MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazoliumbromide) assay, Trans-well migration and Matrigel invasion assay were conducted to study the proliferation, migration and invasion abilities of lung adenocarcinoma cells independently. We found that JAK2 mRNA was up-regulated in lung adenocarcinoma tissues when compared with their adjacent non-tumor tissues, and was associated with lymph node metastasis ( p < 0.05). JAK2 V617F and N30S mutations and JAK2 amplification were detected by NGS in plasmid samples in patients with lung adenocarcinoma. Downregulation of JAK2 inhibited the proliferation, migration and invasion abilities of lung adenocarcinoma A549 cells. Moreover, overexpression of JAK2 induced the proliferation, migration and invasion abilities of A549 cells. Thus, the up-regulation, mutation and amplification of JAK2 detected in lung adenocarcinoma may participate in lung cancer progression by regulating cancer cells' proliferation, migration and invasion.

Suppressing the molecular signaling pathways involved in inflammation and cancer in breast cancer cell lines MDA-MB-231 and MCF-7 by miR-590.

Breast cancer is the most frequent cancer among women worldwide. Tumor immunology suggests relationships between the immune system, chronic inflammation, and cancer. The immune system may either prevent or promote carcinogenesis. Here, we evaluated molecular signaling pathways common in inflammation and cancer and detected the microRNAs which play pivotal roles in mediating these pathways. Using bioinformatics assays, signaling pathways common in inflammation and cancer, and microRNAs mediating these pathways were identified. MiR-590 was selected and cloned into the pLenti-III-eGFP vector and transfected into the breast cancer cell lines. The expression level of microRNA and the candidate genes was evaluated by real-time quantitative reverse transcription polymerase chain reaction, and the apoptosis level in transfected cells was measured by Annexin V-7AAD assay. The cell migration was tested by real-time quantitative reverse transcription polymerase chain reaction for MMP2/MMP9. The expression levels of miR-590 and the selected genes (i.e. JAK2, PI3K, MAPK1, and CREB) were measured 72 h after transfection. While miR-590 showed an over-expression, the genes were significantly down-regulated. A significant increase was observed in apoptosis level in both cell lines and MMP2/MMP9 was significantly decreased in MDA-MB-231 cells. MiR-590 was selected as a microRNA which triggers and down-regulates critical genes of signaling pathways similar in cancer and inflammation. Following the miR-590 treatment, JAK2, PI3K, MAPK1, and CREB were down-regulated and the apoptosis level was increased in breast cancer cell lines. Apparently, some microRNAs can be good candidates for novel treatments of cancer. Although miR-590 showed good results in this study, further studies are required to investigate the role of miR-590 in breast cancer therapy.

Rankl Impairs Lactogenic Differentiation Through Inhibition of the Prolactin/Stat5 Pathway at Midgestation.

Prolactin and progesterone both orchestrate the proliferation and differentiation of the mammary gland during gestation. Differentiation of milk secreting alveoli depends on the presence of prolactin receptor, the downstream Jak2-Stat5 pathway and the transcription factor Elf5. A strict regulation of Rank signaling is essential for the differentiation of the mammary gland and in particular for alveolar commitment. Impaired alveologenesis and lactation failure are observed in both, knockout and Rank overexpressing mice; however, the underlying molecular mechanism responsible for these phenotypes remains largely unknown. Using genome-wide expression analyses and functional studies, we show here that Rankl (RL) exposure leads to impaired secretory differentiation of alveolar cells not only in MMTV-RANK but also in wild-type (WT) mammary acini. Conversely, pharmacological blockage of Rank signaling at midgestation in WT mice leads to precocious and exacerbated lactogenesis. Mechanistically, RL negatively regulates Stat5 phosphorylation and Elf5 expression at the onset of lactogenesis. Continuous RL exposure leads to the expansion of basal and bipotent cells in WT and MMTV-RANK acini. Overall, we demonstrate that enhanced Rank signaling impairs secretory differentiation during pregnancy by inhibition of the prolactin/p-Stat5 pathway.

Overexpression of JAK2: a predictor of unfavorable prognosis for nasopharyngeal carcinoma.

PURPOSE: Analysis of the nasopharyngeal carcinoma public transcriptome revealed JAK2 was significantly upregulated in tumors, which encouraged us to investigate its prognostic significance and mutational status. MATERIALS & METHODS: We assessed the immune-expression of JAK2 and its relationships with various clinicopathological parameters. JAK2 mutation was detected by PCR followed by sequencing. RESULTS: High expression of JAK2 was significantly associated with advanced tumor staging (p = 0.019). JAK2 overexpression acted as an independent predictor for worse disease-specific survival (p = 0.005), distant metastasis-free survival (p = 0.036), local recurrence-free survival (p = 0.012) and overall survival (p = 0.007). JAK2 mutation was not detected in selected cases with JAK2 protein overexpression. CONCLUSION: JAK2 can serve as a valuable negative prognostic factor and a potential therapeutic target.

miR-204 suppresses non-small-cell lung carcinoma (NSCLC) invasion and migration by targeting JAK2.

Aberrant expression of microRNA is associated with the development and progression of cancers. MicroRNA-204 (miR-204) down-regulation has been previously demonstrated in non-small-cell lung carcinoma (NSCLC); however, the underlying mechanism by which miR-204 suppresses tumorigenesis in NSCLC remains elusive. In this study, miR-204 expression was found to be down-regulated, and that of Janus kinase 2 (JAK2) was found to be up-regulated in four NSCLC cell lines (A549, H1299, H1650, and H358) compared to the normal lung cell line. The overexpression of miR-204 suppressed the invasive and migratory capacities of H1299 cells. A luciferase assay confirmed that the binding of miR-124 to the -untranslated region of JAK2 inhibited the expression of JAK2 proteins in H1299 cells. JAK-2 overexpression effectively reversed miR-204-repressed NSCLC metastasis. Taken together, our findings revealed that miR-204 functions as a tumor suppressor in NSCLC by targeting JAK2, and that miR-204 may therefore serve as a biomarker for the diagnosis and treatment of NSCLC.

Higher proliferation of peritumoral endothelial cells to IL-6/sIL-6R than tumoral endothelial cells in hepatocellular carcinoma.

BACKGROUND: This study aimed to explore the responses to the interleukin-6 (IL-6)/soluble interleukin-6 receptor (sIL-6R) complex in peritumoral endothelial cells (PECs) and tumor endothelial cells (TECs), as well as determine the signaling pathways in the angiogenesis of hepatocellular carcinoma (HCC). METHODS: The expression of IL-6, IL-6R, gp130, CD68, HIF-1α, and microvessel density (MVD) were assessed with an orthotopic xenograft model in nude mice. ECs were incubated under hypoxic conditions to detect IL-6 and gp130. The proliferation of PECs and TECs in the presence of IL-6 and sIL-6R, as well as the expression of gp130, JAK2/STAT3, PI3K/AKT in endothelial cells were measured. RESULTS: Peritumoral IL-6, IL-6R, gp130, CD68, and HIF-1α expression, as well as MVD, gradually increased during tumor growth. Hypoxia could directly induce IL-6 expression, but not gp130 in PECs. The co-culture of IL-6/sIL-6R induced much higher PEC proliferation and gp130 expression, as well as the elevated phosphorylation of JAK2 and STAT3, however not the phosphorylation of PI3K and AKT. CONCLUSIONS: PECs exhibited higher proliferation in response to IL-6/sIL-6R co-treatment compared with TECs in HCC via the up-regulation of gp130 /JAK2/STAT3. PEC and its associated peritumoral angiogenesis microenvironment may be a potential novel target for anti-angiogenic treatment.

Jak2/Stat1 pathway mediated tetrahydrobiopterin up-regulation contributes to nitric oxide overproduction in high-glucose cultured rat mesangial cells.

Nitric oxide (NO) is crucial for the progression of early diabetic nephropathy (DN). It is important to clarify the mechanism for the production of NO in mesangial cells (MCs). In this study, the amounts/activities of related factors such as reactive oxygen species (ROS), NO, 3 isoforms of nitric oxide synthase (NOS), tetrahydrobiopterin (BH4), GTP cyclohydrolase I (GTPCH I), Jak2, and Stat1 were determined using high-glucose cultured rat MCs. The results showed that the production of BH4 under oxidative stress was strongly stimulated by its rate-limiting enzyme GTP cyclohydrolase, which increased the expression and activity of inducible NOS to facilitate NO synthesis. Furthermore, the relative quantities of activated-Jak2 and activated-Stat1 were increased. Therefore, Jak2/Stat1 pathway mediated BH4 up-regulation can contribute to excessive NO in high-glucose cultured MCs. Our results will be helpful for screening new targets to improve the therapy for early DN.

Upregulation of the large conductance voltage- and Ca2+-activated K+ channels by Janus kinase 2.

The iberiotoxin-sensitive large conductance voltage- and Ca(2+)-activated potassium (BK) channels (maxi-K(+)-channels) hyperpolarize the cell membrane thus supporting Ca(2+) entry through Ca(2+)-release activated Ca(2+) channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK channels, cRNA encoding Ca(2+)-insensitive BK channels (BK(M513I+Δ899-903)) was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, gain-of-function (V617F)JAK2, or inactive (K882E)JAK2. K(+) conductance was determined by dual electrode voltage clamp and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells, iberiotoxin-sensitive K(+) current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript, and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result, the K(+) current in BK(M513I+Δ899-903)-expressing oocytes was significantly increased following coexpression of JAK2 or (V617F)JAK2 but not (K882E)JAK2. Coexpression of the BK channel with (V617F)JAK2 but not (K882E)JAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel and (V617F)JAK2-expressing oocytes to the JAK2 inhibitor AG490 (40 µM) significantly decreased K(+) current. Inhibition of channel insertion by brefeldin A (5 µM) decreased the K(+) current to a similar extent in oocytes expressing the BK channel alone and in oocytes expressing the BK channel and (V617F)JAK2. The iberiotoxin (50 nM)-sensitive K(+) current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 µM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK channel mRNA and protein abundance. In conclusion, JAK2 upregulates BK channels by increasing channel protein abundance in the cell membrane.

2-Methoxystypandrone inhibits signal transducer and activator of transcription 3 and nuclear factor-κB signaling by inhibiting Janus kinase 2 and IκB kinase.

Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) or the nuclear factor-κB (NF-κB) pathway occurs frequently in cancer cells and contributes to oncogenesis. The activation of Janus kinase 2 (JAK2) and IκB kinase (IKK) are key events in STAT3 and NF-κB signaling, respectively. We have identified 2-methoxystypandrone (2-MS) from a traditional Chinese medicinal herb Polygonum cuspidatum as a novel dual inhibitor of JAK2 and IKK. 2-MS inhibits both interleukin-6-induced and constitutively-activated STAT3, as well as tumor necrosis factor-α-induced NF-κB activation. 2-MS specifically inhibits JAK and IKKß kinase activities but has little effect on activities of other kinases tested. The inhibitory effects of 2-MS on STAT3 and NF-κB signaling can be eliminated by DTT or glutathione and can last for 4 h after a pulse treatment. Furthermore, 2-MS inhibits growth and induces death of tumor cells, particularly those with constitutively-activated STAT3 or NF-κB signaling. We propose that the natural compound 2-MS, as a potent dual inhibitor of STAT3 and NF-κB pathways, is a promising anticancer drug candidate.

The inactivation of JAK2/STAT3 signaling and desensitization of M1 mAChR in minimal hepatic encephalopathy (MHE) and the protection of naringin against MHE.

BACKGROUND: We previously reported that elevation of intracranial dopamine (DA) levels from cirrhotic livers is implicated in the pathogenesis of minimal hepatic encephalopathy (MHE). Intracellular events in neurons, which lead to memory loss in MHE by elevated DA, however, remain elusive. METHODS: In our present study, an MHE rat model, a DA - intracerebroventricularly (i.c.v.) injected rat model and DA-treated primary cortical neurons (PCNs) were used to study this issue using behavioral tests, double-labeled fluorescent staining, immunoblotting, and semi-quantitative RT-PCR. RESULTS: Cognitive impairment was observed in MHE rats and DA (10 µg, i.c.v.)-treated rats. The levels of DA in the cerebral cortex of both MHE and DA (10 µg)-treated rats were increased. DA conversely modulated the p-JAK2/p-STAT3 levels in PCNs. In accordance, DA downregulated an anacetylcholine-producing enzyme, choline acetyltransferase (ChAT), and desensitized the M1-type muscarinic acetylcholine receptor (M1 mAChR). Furthermore, naringin completely restored cognitive function in MHE/DA (10 µg)-treated models by activating the JAK2/STAT3 axis, paralleling the upregulation of ChAT and sensitization of M1 mAChR. CONCLUSIONS: We propose a hypothesis accounting for memory impairment related to MHE: DA-dependent inactivation of the JAK2/STAT3 axis causes memory loss through cholinergic dysfunction. Our findings provide not only a novel pathological hallmark in MHE but also a novel target in MHE therapy.

A highly specific q-RT-PCR assay to address the relevance of the JAK2WT and JAK2V617F expression levels and control genes in Ph-negative myeloproliferative neoplasms.

In Ph- myeloproliferative neoplasms, the quantification of the JAK2V617F transcripts may provide some advantages over the DNA allele burden determination. We developed a q-RT-PCR to assess the JAK2WT and JAK2V617F mRNA expression in 105 cases (23 donors, 13 secondary polycythemia, 22 polycythemia vera (PV), 38 essential thrombocythemia (ET), and 9 primary myelofibrosis (PMF)). Compared with the standard allele-specific oligonucleotide (ASO)-PCR technique, our assay showed a 100 % concordance rate detecting the JAK2V617F mutation in 22/22 PV (100 %), 29/38 (76.3 %) ET, and 5/9 (55.5 %) PMF cases, respectively. The sensitivity of the assay was 0.01 %. Comparing DNA and RNA samples, we found that the JAK2V617F mutational ratios were significantly higher at the RNA level both in PV (p = 0.005) and ET (p = 0.001) samples. In PV patients, JAK2WT expression levels positively correlated with the platelets (PLTs) (p = 0.003) whereas a trend to negative correlation was observed with the Hb levels (p = 0.051). JAK2V617F-positive cases showed the lowest JAK2WT and ABL1 mRNA expression levels. In all the samples, the expression pattern of beta-glucoronidase (GUSB) was more homogeneous than that of ABL1 or ß2 microglobulin (B2M). Using GUSB as normalizator gene, a significant increase of the JAK2V617F mRNA levels was seen in two ET patients at time of progression to PV. In conclusion, the proposed q-RT-PCR is a sensitive and accurate method to quantify the JAK2 mutational status that can also show clinical correlations suggesting the impact of the residual amount of the JAK2WT allele on the Ph- MPN disease phenotype. Our observations also preclude the use of ABL1 as a housekeeping gene for these neoplasms.

Impaired renal growth hormone JAK/STAT5 signaling in chronic kidney disease.

BACKGROUND: Treatment with recombinant human growth hormone (GH) is the standard therapy for short stature in children with chronic kidney disease (CKD). However, concerns have been raised on the potential renal fibrogenic effects of GH. There is no information regarding the renal GH receptor (GHR)-JAK-STAT signaling pathway in CKD. METHODS: Subtotal nephrectomized (CKD) and pair-fed sham-operated control (C) juvenile rats were treated with subcutaneous GH or saline for 2 weeks. A single intravenous GH bolus or vehicle was provided prior to euthanasia. RESULTS: Reduced body weight in CKD was improved with GH therapy. The remnant kidney showed glomerular hypertrophy and early interstitial fibrosis without inflammatory infiltration. Treatment of CKD rats with GH did not worsen renal function or fibrosis. Kidney GHR mRNA and protein levels were reduced and basal phosphorylation of JAK2 and STAT5 was significantly impaired. However, intravenous GH administration prior to sacrifice normalized STAT5 phosphorylation. Basal renal IL6 mRNA and phosphorylation of its downstream signaling molecule STAT3 were increased as was the product of its action, the suppressor of cytokine signaling 3 (SOCS3) mRNA. CONCLUSIONS: Despite known unaltered circulating GH levels, remnant kidneys of uremic growth retarded juvenile rats show impaired basal signaling along the GH-activated JAK2/STAT5 signaling pathway. This may well be a consequence of the reduced GHR level and the inhibitory effect of the increase in IL-6-mediated SOCS3 expression. This renal GH insensitivity, if present in humans, may protect against the potential adverse renal effects of GH administration in CKD patients.