Search

Clinical and experimental data have shown that the receptor for advanced glycation end products (RAGE) is implicated in the pathogenesis of respiratory disorders. In this study, we genotyped five widely-evaluated variants in RAGE gene, aiming to assess their association with the risk for chronic obstructive pulmonary disease (COPD) and asthma in northern Han Chinese. Genotypes were determined in 105 COPD patients, 242 asthma patients and 527 controls. In single-locus analysis, there was significant difference in the genotype distributions of rs1800624 between COPD patients and controls (p=0.022), and the genotype and allele distributions of rs1800625 differed significantly (p=0.040 and 0.016) between asthma patients and controls. Haplotype analysis revealed that haplotype T-A-G-T (allele order: rs1800625, rs1800624, rs2070600, rs184003) was significantly associated with a reduced COPD risk (OR=0.32, 95% CI: 0.06-0.60), and haplotype T-A-A-G was significantly associated with a reduced asthma risk (OR=0.19, 95% CI: 0.04-0.96). Further haplotype-phenotype analysis showed that high- and low-density lipoprotein cholesterol and blood urea nitrogen were significant mediators for COPD (psim=0.041, 0.043 and 0.030, respectively), and total cholesterol was a significant mediator for asthma (psim=0.009). Taken together, our findings indicate that RAGE gene is a promising candidate for COPD and asthma, and importantly both disorders are genetically heterogeneous.

Heat shock factors regulate responses to high temperature, salinity, water deprivation, or heavy metals. Their function in combinations of stresses is, however, not known. Arabidopsis HEAT SHOCK FACTOR A4A (HSFA4A) was previously reported to regulate responses to salt and oxidative stresses. Here we show, that the HSFA4A gene is induced by salt, elevated temperature, and a combination of these conditions. Fast translocation of HSFA4A tagged with yellow fluorescent protein from cytosol to nuclei takes place in salt-treated cells. HSFA4A can be phosphorylated not only by mitogen-activated protein (MAP) kinases MPK3 and MPK6 but also by MPK4, and Ser309 is the dominant MAP kinase phosphorylation site. In vivo data suggest that HSFA4A can be the substrate of other kinases as well. Changing Ser309 to Asp or Ala alters intramolecular multimerization. Chromatin immunoprecipitation assays confirmed binding of HSFA4A to promoters of target genes encoding the small heat shock protein HSP17.6A and transcription factors WRKY30 and ZAT12. HSFA4A overexpression enhanced tolerance to individually and simultaneously applied heat and salt stresses through reduction of oxidative damage. Our results suggest that this heat shock factor is a component of a complex stress regulatory pathway, connecting upstream signals mediated by MAP kinases MPK3/6 and MPK4 with transcription regulation of a set of stress-induced target genes., Heat shock factor A4A is phosphorylated by MPK4; it directly binds to promoters of various chaperon and transcription factor genes and controls responses to combined salt and heat stresses.

Combination chemotherapy is a potentially promising approach to enhance anticancer activity, overcome drug resistance, and improve disease-free and overall survival. The current study investigates the antitumor activity of sodium dichloroacetic acid (DCA) in combination with SIRT2 inhibitor Sirtinol and AGK2. We found that combining DCA with Sirtinol produced a synergistic therapeutic benefit in A549 and H1299 NSCLC cells in vitro and in a mouse A549 xenograft model. Synergistic potentiation of oxidative phosphorylation (OXPHOS) was observed, including decreased glucose consumption, decreased lactate production, increased OCR and increased ROS generation, possibly via co-targeting pyruvate dehydrogenase alpha 1(PDHA1). Mechanically, AGK2 and Sirtinol were found to increase the lysine-acetylation and decrease the serine-phosphorylation of PDHA1, which enabled the two inhibitors to synergize with DCA to further activate PDHA1. Besides, a AMPKα-ROS feed-forward loop was notably activated after the combined treatments compared with mono-therapy. Our results indicate that the combination of DCA and SIRT2 inhibitor may provide a promising therapeutic strategy to effectively kill cancer cells.

Macrophage migration plays an essential role in immune system and is also involved in many pathological situations. However, the regulatory mechanism of macrophage migration remains to be elucidated due to its diverse responses to various stimuli. SAK-HV, a multifunctional protein possessing thrombolytic and lipid-lowering activity, can selectively induce the macrophage proliferation. Here, we reported SAK-HV significantly triggered RAW264.7 cells migration through its functional domain of SAK-mutant by activating both c-jun N-terminal kinases (JNK) and nuclear factor-κB (NF-κB) pathways. Meanwhile, SAK-HV upregulated the expression of some effector proteins, among which only the expression of Monocyte chemoattractant protein-1 (MCP-1) was inhibited by the blockade of JNK and NF-κB pathways. Further research showed that MCP-1 promoted migration ultimately by interacting with Chemokine (C-C motif) Receptor 2 (CCR2) in an autocrine manner. In summary, SAK-HV induced RAW264.7 cells migration through its SAK-mutant domain, during which MCP-1 chemokine mediated by JNK and NF-κB pathways played a key role. These results revealed a novel effect of SAK-HV on modulating macrophage migration and also deepened the understanding of its pharmacodynamics.

BACKGROUND AND PURPOSE: IL‐1β is a cytokine of critical importance in inflammatory, infectious and autoimmune diseases. Zinc finger protein 91 (ZFP91) has been reported to be involved in multiple biological processes. Here, we identified a previously unknown role for ZFP91 in the production of biologically active IL‐1β and investigated the underlying mechanisms of its effects. EXPERIMENTAL APPROACH: In vitro, the underlying mechanisms of ZFP91 at inhibiting the expression of IL‐1β were investigated by ELISA, RT‐PCR, Western blotting, immunoprecipitation and immunofluorescence assays. In vivo, colitis was induced by giving 4% dextran sulfate sodium (DSS) p.o. in drinking water for 5 days. Peritonitis was induced by injecting 700 μg alum i.p. for 12 h. KEY RESULTS: ZFP91 activated the non‐canonical caspase‐8 inflammasome, which resulted in robust IL‐1β secretion. Using an immunoprecipitation assay and immunofluorescence assay, we found that ZFP91 promoted the assembly of the non‐canonical caspase‐8 inflammasome complex. Moreover, ZFP91 enhanced the activation of ERK, p38 MAPK and JNK in macrophages. In addition, our data demonstrate that the synthesis of pro‐IL‐1β is dependent on activation of these MAPK signalling pathways. In vivo experiments, the symptoms and colonic inflammation associated with DSS‐induced colitis were ameliorated in mice deficient in ZFP91. Furthermore, the inflammation in alum‐induced peritonitis was also attenuated in mice deficient in ZFP91. CONCLUSIONS AND IMPLICATIONS: Our research describes a mechanism by which ZFP91 promotes production of IL‐1β under physiological conditions and suggests that ZFP91 may be a promising therapeutic target for intervention in inflammatory, infectious and autoimmune‐related diseases.

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis-caused bone destruction, results from an increase of bone-resorbing osteoclasts (OCs) induced by inflammation. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated that the effect of urokinase-type plasminogen activator (uPA) on inflammatory osteoclastogenesis induced by lipopolysaccharide (LPS), which is a potent stimulator of bone resorption in inflammatory diseases. We found that the uPA deficiency promoted inflammatory osteoclastogenesis and bone loss induced by LPS. We also showed that LPS induced the expression of uPA, and the uPA treatment attenuated the LPS-induced inflammatory osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells. Additionally, we showed that the uPA-attenuated inflammatory osteoclastgenesis is associated with the activation of plasmin/protease-activated receptor (PAR)-1 axis by uPA. Moreover, we examined the mechanism underlying the effect of uPA on inflammatory osteoclastogenesis, and found that uPA/plasmin/PAR-1 activated the adenosine monophosphate-activated protein kinase (AMPK) pathway through Ca2+/calmodulin dependent protein kinase kinase (CaMKK) activation, and attenuated inflammatory osteoclastogenesis by inactivation of NF-κB in RAW264.7 cells. These data suggest that uPA attenuated inflammatory osteoclastogenesis through the plasmin/PAR-1/Ca2+/CaMKK/AMPK axis. Our findings may provide a novel therapeutic approach to bone loss caused by inflammatory diseases.

Oxidative damage and inflammation are closely associated with the pathogenesis of acute lung injury (ALI). Thus, we explored the protective effect of isovitexin (IV), a glycosylflavonoid, in the context of ALI. To accomplish this, we created in vitro and in vivo models by respectively exposing macrophages to lipopolysaccharide (LPS) and using LPS to induce ALI in mice. In vitro, our results showed that IV treatment reduced LPS-induced pro-inflammatory cytokine secretion, iNOS and COX-2 expression and decreased the generation of ROS. Consistent findings were obtained in vivo. Additionally, IV inhibited H2O2-induced cytotoxicity and apoptosis. However, these effects were partially reversed following the use of an HO-1 inhibitor in vitro. Further studies revealed that IV significantly inhibited MAPK phosphorylation, reduced NF-κB nuclear translocation, and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. In vivo, pretreatment with IV attenuated histopathological changes, infiltration of polymorphonuclear granulocytes and endothelial activation, decreased the expression of ICAM-1 and VCAM-1, reduced the levels of MPO and MDA, and increased the content of GSH and SOD in ALI. Furthermore, IV treatment effectively increased Nrf2 and HO-1 expression in lung tissues. Therefore, IV may offer a protective role against LPS-induced ALI by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways.

Cancer cells rely on hyperactive de novo lipid synthesis for maintaining malignancy. Recent studies suggest involvement in cancer of fatty acid oxidation, a process functionally opposite to lipogenesis. A mechanistic link from lipid catabolism to oncogenic processes is yet to be established. Carnitine palmitoyltransferase 1 (CPT1) is a rate-limiting enzyme of fatty acid β-oxidation (FAO) that catalyzes the transfer of long-chain acyl group of the acyl-CoA ester to carnitine, thereby shuttling fatty acids into the mitochondrial matrix for β-oxidation. In the present study, we demonstrated that CPT1A was highly expressed in most ovarian cancer cell lines and primary ovarian serous carcinomas. Overexpression of CPT1A correlated with a poor overall survival of ovarian cancer patients. Inactivation of CPT1A decreased cellular ATP levels and induced cell cycle arrest at G0/G1, suggesting that ovarian cancer cells depend on or are addicted to CPT1A-mediated FAO for cell cycle progression. CPT1A deficiency also suppressed anchorage-independent growth and formation of xenografts from ovarian cancer cell lines. The cyclin-dependent kinase inhibitor p21WAF1 (p21) was identified as most consistently and robustly induced cell cycle regulator upon inactivation of CPT1A. Furthermore, p21 was transcriptionally upregulated by the FoxO transcription factors, which were in turn phosphorylated and activated by AMP-activated protein kinase and the mitogen-activated protein kinases JNK and p38. Our results established the oncogenic relevance of CPT1A and a mechanistic link from lipid catabolism to cell cycle regulation, suggesting that CPT1A could be a prognostic biomarker and rational target for therapeutic intervention of cancer.

// Ahmed Abdelbaset-Ismail 1 , Sylwia Borkowska 2 , Anna Janowska-Wieczorek 3 , Torsten Tonn 4 , Cesar Rodriguez 1 , Marcin Moniuszko 5 , Lukasz Bolkun 7 , Janusz Koloczko 7 , Andrzej Eljaszewicz 5 , Janina Ratajczak 1 , Mariusz Z. Ratajczak 1, 6 , Magda Kucia 1, 6 1 Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, KY, USA 2 Pomeranian Medical University, Szczecin, Poland 3 Department of Hematology, University of Alberta, Edmonton, Canada 4 Transfusion Medicine, Medical Faculty Carl Gustav Carus - Technische Universtitat Dresden, German Red Cross Blood Donation Service North East, Dresden, Germany 5 Department of Regenerative Medicine and Immune Regulation, Medical University of Bialystok, Bialystok, Poland 6 Department of Regenerative Medicine Medical University of Warsaw, Warsaw, Poland 7 Department of Hematology, Medical University of Bialystok, Bialystok, Poland Correspondence to: Mariusz Ratajczak, e-mail: mzrata01@louisville.edu Magda Kucia, e-mail: mjkuci01@louisville.edu Keywords: leukemia, lymphoma, germ line, FSH, LH Received: August 25, 2015 Accepted: November 21, 2015 Published: December 20, 2015 ABSTRACT We recently reported that normal hematopoietic stem cells express functional pituitary sex hormone (SexH) receptors. Here we report for the first time that pituitary-secreted gonadotrophins stimulate migration, adhesion, and proliferation of several human myeloid and lymphoid leukemia cell lines. Similar effects were observed after stimulation of human leukemic cell lines by gonadal SexHs. This effect seems to be direct, as the SexH receptors expressed by leukemic cells responded to stimulation by phosphorylation of MAPK p42/44 and AKT ser473 . Furthermore, in parallel studies we confirmed that human primary patient-derived AML and CML blasts also express several functional SexH receptors. These results shed more light on the potential role of SexHs in leukemogenesis and, in addition, provide further evidence suggesting a developmental link between hematopoiesis and the germline.

Dysregulation of miRNAs has been shown to contribute to the carcinogenesis and progression of nasopharyngeal carcinoma (NPC). Our previous microarray data showed that miR-16 expression is significantly decreased in archived NPC tissues. Here, we confirmed that miR-16 was reduced in NPC cell lines and freshly-frozen samples. Ectopic expression of miR-16 suppressed NPC cell proliferation, migration, and invasion in vitro and inhibited tumor growth and metastatic colonization in the lung in vivo. Furthermore, fibroblast growth factor 2 (FGF2) was identified as a direct target of miR-16, and both phosphoinositide-3- kinase/AKT (PI3K/AKT) and mitogen-activated protein kinase (MAPK) signaling pathways were repressed after miR-16 overexpression. In addition, the restoration of FGF2 reversed the suppressive effects of miR-16. Together, these results indicated that miR-16 suppresses NPC carcinogenesis and progression by targeting FGF2, thereby representing a potential target for miRNA-based therapy for NPC in the future.

The mechanistic target of rapamycin (mTOR) is a central cell growth controller and forms two distinct complexes: mTORC1 and mTORC2. mTORC1 integrates a wide range of upstream signals, both positive and negative, to regulate cell growth. Although mTORC1 activation by positive signals, such as growth factors and nutrients, has been extensively investigated, the mechanism of mTORC1 regulation by stress signals is less understood. In this study, we identified the Nemo-like kinase (NLK) as an mTORC1 regulator in mediating the osmotic and oxidative stress signals. NLK inhibits mTORC1 lysosomal localization and thereby suppresses mTORC1 activation. Mechanistically, NLK phosphorylates Raptor on S863 to disrupt its interaction with the Rag GTPase, which is important for mTORC1 lysosomal recruitment. Cells with Nlk deletion or knock-in of the Raptor S863 phosphorylation mutants are defective in the rapid mTORC1 inhibition upon osmotic stress. Our study reveals a function of NLK in stress-induced mTORC1 modulation and the underlying biochemical mechanism of NLK in mTORC1 inhibition in stress response.

// Yinglong Zhang 1, * , Qiong Ma 1, * , Tao Liu 1, * , Guofeng Guan 2, * , Kailiang Zhang 3 , Jiayan Chen 4 , Nan Jia 1 , Shiju Yan 1 , Guanyin Chen 1 , Shiluan Liu 1 , Kuo Jiang 1 , Yao Lu 1 , Yanhua Wen 1 , Haien Zhao 1 , Yong Zhou 1 , Qingyu Fan 1 , Xiuchun Qiu 1 1 Orthopaedic Oncology Institute, Tangdu Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China 2 Department of Microsurgery, Affiliated Hospital of Binzhou Medical University, Binzhou, Shandong, China 3 Department of Orthopedics, No. 88 Hospital of PLA, Tai’an, Shandong, China 4 Department of Occupational and Environmental Health, School of Public Health, Fourth Military Medical University, Xi’an, Shaanxi, China * These authors have contributed equally to this work Correspondence to: Xiuchun Qiu, e-mail: xiuchun_qiu@yeah.net Qingyu Fan, e-mail: qingyu_fan@yeah.net Keywords: osteosarcoma, tumour self-seeding by circulating tumour cells, interleukin-6, metastasis, recurrence Received: June 15, 2015 Accepted: October 29, 2015 Published: November 09, 2015 ABSTRACT Tumour self-seeding by circulating tumour cells (CTCs) enhances tumour progression and recurrence. Previously, we demonstrated that tumour self-seeding by CTCs occurs in osteosarcoma and revealed that interleukin-6 (IL-6) may promote CTC attraction. Here, we investigated the underlying mechanisms of IL-6 in tumour self-seeding by CTCs. IL-6 suppression inhibited in vitro cell proliferation, migration, and invasion. In addition, rhIL-6 activated the Janus-activated kinase/signal transducers and activators of transcription 3 (JAK/STAT3) and mitogen-activated protein kinase/extracellular-signal regulated kinase1/2 (MAPK/ERK1/2) pathways in vitro . Both pathways increased cell proliferation, but only the JAK/STAT3 pathway promoted migration. Suppressing IL-6 inhibited in vivo tumour growth and metastasis. IL-6 suppression or JAK/STAT3 pathway inhibition reduced CTC seeding in primary tumours. Collectively, IL-6 promotes tumour self-seeding by CTCs in a nude mouse model. This finding may provide a novel strategy for future therapeutic interventions to prevent osteosarcoma progression and recurrence.

// Renata Duchnowska 1 , Piotr J. Wysocki 2 , Konstanty Korski 3 , Bogumila Czartoryska-Arlukowicz 4 , Anna Niwinska 5 , Marlena Orlikowska 6 , Barbara Radecka 7 , Maciej Studzinski 8 , Regina Demlova 9 , Barbara Ziolkowska 10 , Monika Merdalska 11 , Łukasz Hajac 12 , Paulina Myśliwiec 13 , Dorota Zuziak 14 , Sylwia Debska-Szmich 15 , Istvan Lang 16 , Malgorzata Foszczynska-Kloda 2 , Bozenna Karczmarek-Borowska 17 , Anton Żawrocki 18 , Anna Kowalczyk 18 , Wojciech Biernat 18 , Jacek Jassem 18 , for the Central and East European Oncology Group (CEEOG) 1 Military Institute of Medicine, Oncology Department, Warsaw, Poland 2 West Pomeranian Cancer Center, Szczecin, Poland 3 Greater Poland Cancer Center, Poznan, Poland 4 Bialystok Oncology Center, Bialystok, Poland 5 The Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland 6 Warmia and Masuria Oncology Center, Olsztyn, Poland 7 Opole Oncology Center, Poland 8 Oncology Center, Bydgoszcz, Poland 9 Masaryk Memorial Cancer Institute, Brno, Czech Republic 10 Chemotherapy Department, Regional Hospital, Wroclaw, Poland 11 Oncology Center, Kielce, Poland 12 Oncology Center, Wroclaw, Poland 13 Oncology Center, Zielona Gora, Poland 14 Oncology Center, Bielsko-Biala, Poland 15 Medical University of Łodź, Łodź, Poland 16 Department of Medical Oncology, National Institute of Oncology, Budapest, Hungary 17 Department of Chemotherapy, Subcarpathian Oncology Center, Rzeszow, Poland 18 Medical University of Gdansk, Gdansk, Poland Correspondence to: Renata Duchnowska, e-mail: rdtt@wp.pl Keywords: breast cancer, epidermal growth factor receptor type 2, lapatinib, mTOR, p-MAPK Received: May 19, 2015 Accepted: November 13, 2015 Published: November 24, 2015 ABSTRACT Molecular mechanisms of lapatinib resistance in breast cancer are not well understood. The aim of this study was to correlate expression of selected proteins involved in ErbB family signaling pathways with clinical efficacy of lapatinib. Study group included 270 HER2-positive advanced breast cancer patients treated with lapatinib and capecitabine. Immunohistochemical expression of phosphorylated adenosine monophosphate-activated protein (p-AMPK), mitogen-activated protein kinase (p-MAPK), phospho (p)-p70S6K, cyclin E, phosphatase and tensin homolog were analyzed in primary breast cancer samples. The best discriminative value for progression-free survival (PFS) was established for each biomarker and subjected to multivariate analysis. At least one biomarker was determined in 199 patients. Expression of p-p70S6K was independently associated with longer (HR 0.45; 95% CI: 0.25–0.81; p = 0.009), and cyclin E with shorter PFS (HR 1.83; 95% CI: 1.06–3.14; p = 0.029). Expression of p-MAPK (HR 1.61; 95% CI 1.13–2.29; p = 0.009) and cyclin E (HR 2.99; 95% CI: 1.29–6.94; p = 0.011) was correlated with shorter, and expression of estrogen receptor (HR 0.65; 95% CI 0.43–0.98; p = 0.041) with longer overall survival. Expression of p-AMPK negatively impacted response to treatment (HR 3.31; 95% CI 1.48–7.44; p = 0.004) and disease control (HR 3.07; 95% CI 1.25–7.58; p = 0.015). In conclusion: the efficacy of lapatinib seems to be associated with the activity of downstream signaling pathways – AMPK/mTOR and Ras/Raf/MAPK. Further research is warranted to assess the clinical utility of these data and to determine a potential role of combining lapatinib with MAPK pathway inhibitors.

Statins purportedly exert anti-tumoral effects on breast cancer. However, the biologic mechanisms for these actions are not fully elucidated. The aims of this study were 1) to explore the effects of simvastatin on apoptosis, proliferation as well as PI3K/Akt/mTOR and MAPK/ERK pathway in a window-of-opportunity breast cancer trial; 2) to further confirm findings from the clinical trial by functional studies; 3) to explore the regulatory role of mevalonate pathway on the anti-tumoral effects of simvastatin. In clinical samples, simvastatin led to increase in cleaved caspase-3 (p = 0.002) and decreased trend for Ki67 (p = 0.245). Simvastatin markedly suppressed PI3K/Akt/mTOR signalling by activating PTEN (p = 0.005) and by dephosphorylating Akt (p = 0.002) and S6RP (p = 0.033); it also inhibited MAPK/ERK pathway by dephosphorylating c-Raf (p = 0.018) and ERK1/2 (p = 0.002). In ER-positive (MCF-7, T47D) and ER-negative (MDA-MB-231, BT-549) breast cancer cells, simvastatin treatment consistently induced apoptosis and inhibited proliferation by deregulating caspase cascades and cell cycle proteins in a dose dependent manner. Concordantly, simvastatin strongly suppressed PI3K/Akt/mTOR pathway by enhancing PTEN expression and by further sequentially dephosphorylating downstream cascades including Akt, mTOR, p70S6K, S6RP and 4E-BP1. Furthermore, simvastatin significantly inhibited MAPK/ERK pathway by dephosphorylating sequential cascades such as c-Raf, MEK1/2 and ERK1/2. These simvastatin anti-tumoral effects were reversed by metabolic products of the mevalonate pathway, including mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate. These findings shed light on the biological and potential anti-tumoral effects of simvastatin in breast cancer.

Ovarian cancer is the 5th leading cause of cancer death among women in the United States. The mevalonate pathway is thought to be a potential oncogenic pathway in the pathogenesis of ovarian cancer. Simvastatin, a 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) inhibitor, is a widely used drug for inhibiting the synthesis of cholesterol and may also have anti-tumorigenic activity. Our goal was to evaluate the effects of simvastatin on ovarian cancer cell lines, primary cultures of ovarian cancer cells and in an orthotopic ovarian cancer mouse model. Simvastatin significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, and caused cellular stress via reduction in the enzymatic activity of HMGCR and inhibition of the MAPK and mTOR pathways in ovarian cancer cells. Furthermore, simvastatin induced DNA damage and reduced cell adhesion and invasion. Simvastatin also exerted anti-proliferative effects on primary cell cultures of ovarian cancer. Treatment with simvastatin in an orthotopic mouse model reduced ovarian tumor growth, coincident with decreased Ki-67, HMGCR, phosphorylated-Akt and phosphorylated-p42/44 protein expression. Our findings demonstrate that simvastatin may have therapeutic benefit for ovarian cancer treatment and be worthy of further exploration in clinical trials.

// Haibo Su 1 , Cong Kong 1 , Lin Zhu 1 , Qi Huang 1 , Liulin Luo 1,2 , Honghai Wang 1 and Ying Xu 1 1 State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai, China 2 Department of Clinical Laboratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Correspondence to: Ying Xu, email: // Honghai Wang, email: // Keywords : PPE26, PE/PPE, mycobacterium tuberculosis, TLR2, proteomics, Immunology and Microbiology Section, Immune response and Immunity Received : June 21, 2015 Accepted : September 12, 2015 Published : October 02, 2015 Abstract The pathophysiological functions and the underlying molecular basis of PE /PPE proteins of M. tuberculosis remain largely unknown. In this study, we focused on the link between PPE26 and host response. We demonstrated that PPE26 can induce extensive inflammatory responses in macrophages through triggering the cross-talk of multiple pathways involved in the host response, as revealed by iTRAQ-based subcellular quantitative proteomics. We observed that PPE26 is able to specifically bind to TLR2 leading to the subsequent activation of MAPKs and NF-κB signaling. PPE26 functionally stimulates macrophage activation by augmenting pro-inflammatory cytokine production (TNF-α, IL-6 and IL-12 p40) and the expression of cell surface markers (CD80, CD86, MHC class I and II). We observed that PPE26-treated macrophages effectively polarizes naive CD4 + T cells to up-regulate CXCR3 expression, and to secrete IFN-γ and IL-2, indicating PPE26 contributes to the Th1 polarization during the immune response. Importantly, rBCG::PPE26 induces stronger antigen-specific TNF-α and IFN-γ activity, and higher levels of the Th1 cytokines TNF-α and IFN-γ comparable to BCG. Moreover, PPE26 effectively induces the reciprocal expansion of effector/memory CD4 + /CD8 + CD44 high CD62L low T cells in the spleens of mice immunized with this strain. These results suggest that PPE26 may be a TLR2 agonist that stimulates innate immunity and adaptive immunity, indicating that PPE26 is a potential antigen for the rational design of an efficient vaccine against M. tuberculosis .

Angiosarcomas are rare malignant mesenchymal tumors of endothelial differentiation. The clinical behavior is usually aggressive and the prognosis for patients with advanced disease is poor with no effective therapies. The genetic bases of these tumors have been partially revealed in recent studies reporting genetic alterations such as amplifications of MYC (primarily in radiation-associated angiosarcomas), inactivating mutations in PTPRB and R707Q hotspot mutations of PLCG1. Here, we performed a comprehensive genomic analysis of 34 angiosarcomas using a clinically-approved, hybridization-based targeted next-generation sequencing assay for 341 well-established oncogenes and tumor suppressor genes. Over half of the angiosarcomas (n = 18, 53%) harbored genetic alterations affecting the MAPK pathway, involving mutations in KRAS, HRAS, NRAS, BRAF, MAPK1 and NF1, or amplifications in MAPK1/CRKL, CRAF or BRAF. The most frequently detected genetic aberrations were mutations in TP53 in 12 tumors (35%) and losses of CDKN2A in 9 tumors (26%). MYC amplifications were generally mutually exclusive of TP53 alterations and CDKN2A loss and were identified in 8 tumors (24%), most of which (n = 7, 88%) arose post-irradiation. Previously reported mutations in PTPRB (n = 10, 29%) and one (3%) PLCG1 R707Q mutation were also identified. Our results demonstrate that angiosarcomas are a genetically heterogeneous group of tumors, harboring a wide range of genetic alterations. The high frequency of genetic events affecting the MAPK pathway suggests that targeted therapies inhibiting MAPK signaling may be promising therapeutic avenues in patients with advanced angiosarcomas. OA gold

Epstein-Barr virus (EBV) is an oncogenic gammaherpes virus which is linked to pathogenesis of several human lymphatic malignancies. The EBV essential latent antigen EBNA3C is critical for efficient conversion of primary human B-lymphocytes to lymphoblastic cell lines and for continued LCL growth. EBNA3C, an EBV latent antigen with oncogenic potential can bind and regulate the functions of a wide range of cellular transcription factors. In our current reverse genetics study, we deleted the full length EBNA3C, and independently the RBP-Jκ and Nm23-H1 binding sites within EBNA3C using BACmid recombinant engineering methodology. Our experiments demonstrated that deletion of the EBV EBNA3C open reading frame (ORF) and more specifically the residues 621-675 which binds Nm23H1 and SUMO-1 showed a significant reduction in the ability of the cells to proliferate. Furthermore, they exhibited lower infectivity of human peripheral blood mononuclear cells (PBMCs). We also showed that recombinant EBV with deletions of the EBNA3C ORF, as well as a recombinant with residues 621-675 within EBNA3C ORF deleted had diminished abilities to activate CD40. Our study also revealed that the full length (1-992) and 621-675 aa deletions of EBNA3C when compared to wild type EBV infected PBMCs had differential expression patterns for the phosphorylation of MAP kinases specifically p38, JNK and ERK. Regulation of β-catenin also differed among wild type and EBNA3C deleted mutants. These temporal differences in signaling activities of these recombinant viruses in PBMCs is likely important in defining their functional importance in EBV-mediated B-cell transformation.

Melanoma is the most deadly form of cutaneous malignancy, and its incidence rates are rising worldwide. In melanoma, constitutive activation of the BRAF/MEK/ERK (MAPK) and PI3K/AKT/mTOR (PI3K) signaling pathways plays a pivotal role in cell proliferation, survival and tumorigenesis. A combination of compounds that lead to an optimal blockade of these critical signaling pathways may provide an effective strategy for prevention and treatment of melanoma. The phytochemical fisetin is known to possess anti-proliferative and pro-apoptotic activities. We found that fisetin treatment inhibited PI3K signaling pathway in melanoma cells. Therefore, we investigated the effect of fisetin and sorafenib (an RAF inhibitor) alone and in combination on cell proliferation, apoptosis and tumor growth. Combination treatment (fisetin + sorafenib) more effectively reduced the growth of BRAF-mutated human melanoma cells at lower doses when compared to individual agents. In addition, combination treatment resulted in enhanced (i) apoptosis, (ii) cleavage of caspase-3 and PARP, (iii) expression of Bax and Bak, (iv) inhibition of Bcl2 and Mcl-1, and (v) inhibition of expression of PI3K, phosphorylation of MEK1/2, ERK1/2, AKT and mTOR. In athymic nude mice subcutaneously implanted with melanoma cells (A375 and SK-MEL-28), we found that combination therapy resulted in greater reduction of tumor growth when compared to individual agents. Furthermore, combination therapy was more effective than monotherapy in: (i) inhibition of proliferation and angiogenesis, (ii) induction of apoptosis, and (iii) inhibition of the MAPK and PI3K pathways in xenograft tumors. These data suggest that simultaneous inhibition of both these signaling pathways using combination of fisetin and sorafenib may serve as a therapeutic option for the management of melanoma.

// Sheng-qi Wang 1 , Shi-ting Liu 1 , Bo-xin Zhao 1 , Fu-heng Yang 1 , Ya-tian Wang 1 , Qian-ying Liang 1 , Ya-bin Sun 2 , Yuan Liu 1 , Zhi-hua Song 1 , Yun Cai 1 , Guo-feng Li 1 1 Department of Pharmacy, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China 2 GCP Center, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China Correspondence to: Guo-Feng Li, e-mail: liguofeng_2010@126.com Keywords: tyrosine kinase inhibitor, afatinib, ovarian cancer, multidrug resistance, ABCB1 Received: March 12, 2015 Accepted: July 10, 2015 Published: July 20, 2015 ABSTRACT ABCB1-mediated multidrug resistance (MDR) remains a major obstacle to successful chemotherapy in ovarian cancer. Herein, afatinib at nontoxic concentrations significantly reversed ABCB1-mediated MDR in ovarian cancer cells in vitro ( p < 0.05). Combining paclitaxel and afatinib caused tumor regressions and tumor necrosis in A2780T xenografts in vivo . More interestingly, unlike reversible TKIs, afatinib had a distinctive dual-mode action. Afatinib not only inhibited the efflux function of ABCB1, but also attenuated its expression transcriptionally via down-regulation of PI3K/AKT and MAPK/p38-dependent activation of NF-κB. Furthermore, apart from a substrate binding domain, afatinib could also bind to an ATP binding domain of ABCB1 through forming hydrogen bonds with Gly533, Gly534, Lys536 and Ala560 sites. Importantly, mutations in these four binding sites of ABCB1 and the tyrosine kinase domain of EGFR were not correlated with the reversal activity of afatinib on MDR. Given that afatinib is a clinically approved drug, our results suggest combining afatinib with chemotherapeutic drugs in ovarian cancer. This study can facilitate the rediscovery of superior MDR reversal agents from molecular targeted drugs to provide a more effective and safer way of resensitizing MDR.

Multiple juxtacrine and paracrine interactions occur between cancer cells and non-cancer cells of the tumor microenvironment (TME) that direct tumor progression. Cancer Associated Fibroblasts (CAFs) are an integral component of the TME, and the majority of breast tumor stroma is comprised of CAFs. Heterotypic interactions between cancer cells and non-cancer cells of the TME occur via soluble agents, including cytokines, hormones, growth factors, and secreted microRNAs. We previously identified a microRNA signature indicative of hyperactive MAPK signaling (hMAPK-miRNA signature) that significantly associated with reduced recurrence-free and overall survival. Here we report that the hMAPK-miRNA signature associates with a high metric of stromal cell infiltrate, and we investigate the role of microRNAs, particularly hMAPK-microRNAs, secreted by CAFs on estrogen receptor (ER) expression in breast cancer cells. ER-positive MCF-7/ltE2- cells were treated with conditioned media (CM) from CAFs derived from breast cancers of different PAM50 subtypes (CAFBAS, CAFHER2, and CAFLA). CAF CM isolated specifically from ER-negative primary breast tumors led to ER repression in vitro. Nanoparticle tracking analysis and transmission electron microscopy confirmed the presence of CAF-secreted exosomes in CM and the uptake of these exosomes by the ER+ MCF-7/ltE2- cells. Differentially expressed microRNAs in CAF CM as well as in MCF-7/ltE2- cells treated with this CM were identified. Knockdown of miR-221/222 in CAFBAS resulted in knockdown of miR221/222 levels in the conditioned media and the CM from CAFBAS; miR221/222 knockdown rescued ER repression in ER-positive cell lines treated with CAFBAS-CM. Collectively, our results demonstrate that CAF-secreted microRNAs are directly involved in ER-repression, and may contribute to the MAPK-induced ER repression in breast cancer cells.

Sulforaphane (SFN), a natural compound derived from broccoli/broccoli sprouts, has been demonstrated to be used as an antitumor agent in different types of cancers. However, its antitumor effect in thyroid cancer remains largely unknown. The aim of the study was to investigate the therapeutic potential of SFN for thyroid cancer and explore the mechanisms underlying antitumor effects of SFN by in vitro and in vivo studies. Our data demonstrated that SFN significantly inhibited thyroid cancer cell proliferation in a dose- and time-dependent manner, induced G2/M phase cell cycle arrest and apoptosis, and inhibited thyroid cancer cell migration and invasion by suppressing epithelial-mesenchymal transition (EMT) process and expression of Slug, Twist, MMP-2 and -9. Mechanically, SFN inhibited thyroid cancer cell growth and invasiveness through repressing phosphorylation of Akt, enhancing p21 expression by the activation of Erk and p38 signaling cascades, and promoting mitochondrial-mediated apoptosis via reactive oxygen species (ROS)-dependent pathway. Growth of xenograft tumors derived from thyroid cancer cell line FTC133 in nude mice was also significantly inhibited by SFN. Importantly, we did not find significant effect of SFN on body weight and liver function of mice. Collectively, we for the first time demonstrate that SFN is a potentially effective antitumor agent for thyroid cancer.

The coiled coil is a superhelical structural protein motif involved in a diverse array of biological functions, and the abnormal expression of the coiled-coil domain containing proteins has a direct link with the phenotype of tumor cell migration, invasion and metastasis. The aim of this study was to investigate the critical role of Coiled-coil domain-containing protein 34 (CCDC34) in bladder carcinogenesis, which has never been reported to date. Here, we found CCDC34 expression was elevated in bladder cancer tissues and cell lines. The knockdown of CCDC34 via lentivirus-mediated siRNA significantly suppressed bladder cancer cells proliferation and migration, and induced cell cycle arrest at G2/M phase and increased apoptosis in vitro. In addition, CCDC34 knockdown suppressed bladder tumor growth in nude mice. Moreover, CCDC34 silencing decreased the phosphorylation of MEK, ERK1/2, JNK, p38 and Akt, and the expressions of c-Raf and c-Jun, indicating MAPK and AKT pathways (ERK/MAPK, p38/MAPK, JNK/MAPK and PI3K/Akt) might be involved in CCDC34 regulation of bladder cancer cell proliferation and migration. Our findings revealed for the first time a potential oncogenic role for CCDC34 in bladder carcinoma pathogenesis and it may serve as a biomarker or even a therapeutic target for bladder cancer.

// Xufu Wei 1, 2 , Chengyong Tang 3 , Xiuxian Lu 1 , Rui Liu 1 , Mi Zhou 1 , Diao He 1 , Daofeng Zheng 1 , Chao Sun 1 , Zhongjun Wu 1 1 Department of Hepatobiliary Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China 2 Department of Pathology and Division of Biological Sciences, University of California San Diego, La Jolla, California, USA 3 Department of Clinical Pharmacology, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China Correspondence to: Zhongjun Wu, e-mail: wzjtcy@126.com Keywords: hepatocellular carcinoma, miR-101, macrophage, DUSP1, sorafenib Received: March 14, 2015 Accepted: May 18, 2015 Published: May 29, 2015 ABSTRACT Hepatocellular carcinoma (HCC)-associated macrophages accelerate tumor progression via growth factor release. Therefore, tumor-associated macrophages (TAMs)-initiated signaling cascades are potential therapeutic targets. To better understand anticancer effects of systemic HCC therapy, we studied sorafenib’s effect on macrophage function, focusing on macrophage-related growth factor secretion. We found that dual specificity phosphatase 1 (DUSP1) is a direct target of miR-101. Transfection of miR-101 reduced DUSP1 induction in M2 macrophages and prolonged ERK1/2, p38 and JNK activation, whereas inhibition of miR-101 enhanced DUSP1 expression and decreased ERK1/2, p38 and JNK activation. miR-101 expression was decreased by sorafenib, and inhibition of PI3K/AKT blocked induction of miR-101 by LPS in M2 cells. M2 cells with greater TGF-β and CD206 mRNA expression compared to M1 cells had increased hepatoma growth, metastases and EMT. Sorafenib inhibited miR-101 expression and enhanced DUSP1 expression and lowered TGF-β and CD206 release in M2 cells, slowing macrophage-driven HCC. Our studies demonstrate miR-101 regulates macrophage innate immune responses to LPS via targeting DUSP1. Sorafenib alters macrophage polarization, reduces TGF-β driven cancer growth, metastases and EMT in vitro , and partially inhibits macrophage activation in vivo . Thus, macrophage modulation might explain the anticancer effects of sorafenib.

Glioblastoma (GBM) is the most lethal brain cancer with profound genomic alterations. While the bona fide tumor suppressor genes such as PTEN, NF1, and TP53 have high frequency of inactivating mutations, there may be the genes with GBM-suppressive roles for which genomic mutation is not a primary cause for inactivation. To identify such genes, we employed in vivo RNAi screening approach using the patient-derived GBM xenograft models. We found that Nemo-Like Kinase (NLK) negatively regulates mesenchymal activities, a characteristic of aggressive GBM, in part via inhibition of WNT/β-catenin signaling. Consistent with this, we found that NLK expression is especially low in a subset of GBMs that harbors high WNT/mesenchymal activities. Restoration of NLK inhibited WNT and mesenchymal activities, decreased clonogenic growth and survival, and impeded tumor growth in vivo. These data unravel a tumor suppressive role of NLK and support the feasibility of combining oncogenomics with in vivo RNAi screen.

Lung cancers express non-neuronal, cholinergic autoparacrine loop, which facilitates tumor growth. Interruption of M3 muscarinic cholinergic signaling has been reported to inhibit small cell lung cancer (SCLC) growth. The purpose of this study is to investigate if blocking autoparacrine muscarinic cholinergic signaling could inhibit non-small cell lung cancer (NSCLC) growth and possible underlying mechanisms. Our results showed that PC9 and A549 cells expressed all 5 subtypes of muscarinic receptor (mAChR) and blocking M2 mAChR (M2R) signaling using selective antagonist methoctramine or short hairpin RNA (shRNA) inhibited tumor cell proliferation in vitro and in vivo. Consistent with AChR agonists stimulating p44/42 MAPK (Erk1/2) and Akt phosphorylation, blocking M2R signaling decreased MAPK and Akt phosphorylation, indicating that non-neuronal ACh functions as an autoparacrine growth factor signaling in part through activation of M2R and downstream MAPK and Akt pathways. Importantly, further studies revealed that blocking M2R signaling also reversed epithelial-mesenchymal transition (EMT) in vitro and in vivo, indicating that non-neuronal ACh promotes EMT partially through activation of M2R. These findings demonstrate that M2R plays a role in the growth and progression of NSCLC and suggest M2R antagonists may be an efficacious adjuvant therapy for NSCLC.

A structural element termed ‘hydrophobic core’ is a suppressor of spontaneous autophosphorylation in Hog1 and p38s. Practically any mutation in this core of Hog1, but not of p38, evokes spontaneous autophosphorylation. This inherent autophosphorylation suppressor is tighter in mammalian's p38s., Many eukaryotic protein kinases (EPKs) are autoactivated through autophosphorylation of their activation loop. Mitogen-activated protein (MAP) kinases do not autophosphorylate spontaneously; relying instead upon mitogen-activated protein kinase (MAPK) kinases (MKKs) for their activation loop phosphorylation. Yet, in previous studies we identified mutations in the yeast MAPK high osmolarity glycerol (Hog1) that render it capable of spontaneous autophosphorylation and consequently intrinsically active (MKK-independent). Four of the mutations occurred in hydrophobic residues, residing in the αC-helix, which is conserved in all EPKs, and in the αL16-helix that is unique to MAPKs. These four residues interact together forming a structural element termed ‘hydrophobic core’. A similar element exists in the Hog1’s mammalian orthologues p38s. Here we show that the ‘hydrophobic core’ is a loose suppressor of Hog1’s autophosphorylation. We inserted 18 point mutations into this core, 17 of which were able to render Hog1 MKK-independent. In p38s, however, only a very few mutations in the equivalent residues rendered these proteins intrinsically active. Structural analysis revealed that a salt bridge between the αC-helix and the αL16-helix that exists in p38α may not exist in Hog1. This bond further stabilizes the ‘hydrophobic core’ of p38, making p38 less prone to de-repressing its concealed autophosphorylation. Mutating equivalent hydrophobic residues in Jnk1 and Erk2 has no effect on their autophosphorylation. We propose that specific structural elements developed in the course of evolution to suppress spontaneous autophosphorylation of Hog1/p38. The suppressors were kept wobbly, probably to allow activation by induced autophosphorylation, but became stricter in mammalian p38s than in the yeast Hog1.

Rac1, known as a "molecular switch", plays a crucial role in plenty of cellular processes. Rac1 aggravates the damage of myocardial cells in the process of myocardial ischemia-reperfusion during myocardial infarction through activating the NADPH oxidase and bringing about the reactive oxygen species(ROS) generation. Myocardial ischemia and hypoxia are the basic pathogenesis of myocardial infarction and the underlying mechanisms are intricate and varied. Moreover, the regulatory effect of Rac1 on myocardial cells in the condition of serum starvation and the potential mechanisms are still incompletely undefined. Therefore, heart-derived H9c2 cells cultured in 0% serum were used to mimic ischemic myocardial cells and to clarify the role of Rac1 in H9c2 cells and the underlying mechanisms during serum deficiency. After Rac1 was knocked down using specific siRNA, cell apoptosis was assessed by flow cytometry assay and cell proliferation was detected by CCK-8 assay and EdU assay. In addition, the expression and activation of protein in related signaling pathway were detected by Western blot and siRNAs was used to testify the signaling pathways. Our results indicated that Rac1 inhibited apoptosis, promoted proliferation and cell cycle progression of H9c2 cells during serum deficiency. We concluded that Rac1 inhibited apoptosis in an AKT2/MCL1 dependent way and promoted cell proliferation through JNK/c-JUN/Cyclin-D1.

KEY POINTS: Balloon‐assisted enteroscopy (BAE) is an emerging standard procedure by utilizing distensible balloons to facilitate deep endoscopy in the small and large intestine. Sporadic cases of bacteraemia were found after BAE. Balloon distension by BAE caused gut tissue hypoxia. The impact of balloon distension‐induced hypoxia on intestinal barriers remains unclear. Murine models of BAE by colonic balloon distension showed that short‐ and long‐term hypoxia evoked opposite effects on epithelial tight junctions (TJs). Short‐term hypoxia fortified TJ integrity, whereas long‐term hypoxia caused damage to barrier function. Our data showed for the first time the molecular mechanisms and signalling pathways of epithelial barrier fortification and TJ reorganization by short‐term hypoxia for the maintenance of gut homeostasis. The findings suggest avoiding prolonged balloon distension during BAE to reduce the risk of hypoxia‐induced gut barrier dysfunction. ABSTRACT: Balloon‐assisted enteroscopy (BAE) is an emerging standard procedure that uses distensible balloons to facilitate deep endoscopy. Intestines are known to harbour an abundant microflora. Whether balloon distension causes perturbation of blood flow and gut barrier dysfunction, and elicits risk of bacterial translocation remains unknown. Our aims were to (1) conduct a prospective study to gather microbiological and molecular evidence of bacterial translocation by BAE in patients, (2) establish a murine model of colonic balloon distension to investigate tissue hypoxia and intestinal barrier, and (3) assess the effect of short‐ and long‐term hypoxia on epithelial permeability using cell lines. Thirteen patients were enrolled for BAE procedures, and blood samples were obtained before and after BAE for paired comparison. Four of the 13 patients (30.8%) had positive bacterial DNA in blood after BAE. Post‐BAE endotoxaemia was higher than the pre‐BAE level. Nevertheless, no clinical symptom of sepsis or fever was reported. To mimic clinical BAE, mice were subjected to colonic balloon distension. Local tissue hypoxia was observed during balloon inflation, and reoxygenation after deflation. A trend of increased gut permeability was seen after long‐term distension, whereas a significant reduction of permeability was observed by short‐term distension in the proximal colon. Human colonic epithelial Caco‐2 cells exposed to hypoxia for 5–20 min exhibited increased tight junctional assembly, while those exposed to longer hypoxia displayed barrier disruption. In conclusion, sporadic cases of bacteraemia were found after BAE, without septic symptoms. Short‐term hypoxia by balloon distension yielded a protective effect whereas long‐term hypoxia caused damage to the gut barrier.

Key message In plants, phosphorylated MAPKs display constitutive nuclear localization; however, not all studied plant species show co-localization of activated MAPKs to mitotic microtubules. Abstract The mitogen-activated protein kinase (MAPK) signaling pathway is involved not only in the cellular response to biotic and abiotic stress but also in the regulation of cell cycle and plant development. The role of MAPKs in the formation of a mitotic spindle has been widely studied and the MAPK signaling pathway was found to be indispensable for the unperturbed course of cell division. Here we show cellular localization of activated MAPKs (dually phosphorylated at their TXY motifs) in both interphase and mitotic root meristem cells of Lupinus luteus, Pisum sativum, Vicia faba (Fabaceae) and Lycopersicon esculentum (Solanaceae). Nuclear localization of activated MAPKs has been found in all species. Co-localization of these kinases to mitotic microtubules was most evident in L. esculentum, while only about 50 % of mitotic cells in the root meristems of P. sativum and V. faba displayed activated MAPKs localized to microtubules during mitosis. Unexpectedly, no evident immunofluorescence signals at spindle microtubules and phragmoplast were noted in L. luteus. Considering immunocytochemical analyses and studies on the impact of FR180204 (an inhibitor of animal ERK1/2) on mitotic cells, we hypothesize that MAPKs may not play prominent role in the regulation of microtubule dynamics in all plant species. Electronic supplementary material The online version of this article (doi:10.1007/s00299-015-1752-6) contains supplementary material, which is available to authorized users.

Hypoxia-inducible factor-1 (HIF-1) is one of the most promising pharmacological targets for all types of cancer, including ovarian cancer. Ovarian clear cell carcinoma (OCCC) has poor prognosis because of its insensitivity to chemotherapy. To elucidate the characteristics of this troublesome cancer, we examined HIF-1α expression under normoxia or hypoxia in various ovarian cancer cell lines. HIF-1α was highly expressed under normoxia only in RMG-1, an OCCC cell line. To examine whether HIF-1 is involved in the tumorigenesis of RMG-1 cells, we established HIF-1α-silenced cells, RMG-1HKD. The proliferation rate of RMG-1HKD cells was faster than that of RMG-1 cells. Furthermore, the activity of MEK/ERK in the Ras pathway increased in RMG-1HKD cells, whereas that of mTOR in the PI3K pathway did not change. Activation of the Ras pathway was attributable to the increase in phosphorylated MEK via PP2A inactivation. To confirm the crosstalk between the PI3K and Ras pathways in vivo, RMG-1 or RMG-1HKD cells were transplanted into the skin of nude mice with rapamycin (an inhibitor of mTOR), PD98059 (an inhibitor of MEK), or both. RMG-1HKD cells showed higher sensitivity to PD98059 than that observed in RMD-1 cells, whereas the combination therapy resulted in synergistic inhibition of both cells. These findings suggest that inhibition of HIF-1, a downstream target of mTOR in the PI3K pathway, activates the Ras pathway on account of the increase in MEK phosphorylation via PP2A inactivation, and the crosstalk between the 2 pathways could be applied in the combination therapy for HIF-1-overexpressing cancers such as OCCC.

Highlight Transgenically imposed high glutathione, uncoupled from cellular controls, causes an oxidative shift in cytosolic redox potential, activates tobacco MAPKs, and primes immune responses to adapted and non-adapted Pseudomonas syringae pathogens., The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed.

GhMKK6 and ghr-miR5272a comprise a feedback loop in cotton and ghr-miR5272a-mediated regulation of GhMKK6 expression contributes to the immune response against Fusarium wilt., Fusarium wilt is a major biotic stress affecting the productivity of cotton (Gossypium hirsutum). Although mitogen-activated protein kinase (MAPK) cascades play critical roles in plant disease resistance, their intricate regulation under fungal stress remains unclear, especially with regards to microRNA-mediated regulation of MAPK gene expression. In this study, we report that the MAPK kinase gene GhMKK6 and ghr-miR5272a work together in cotton resistance to Fusarium wilt. Silencing GhMKK6 in cotton decreased resistance to F. oxysporum by repressing the expression of known disease-resistance genes. Furthermore, although GhMKK6 played a positive role in disease resistance, excessive GhMKK6 activation caused an excessive hypersensitive response. ghr-miR5272a, a major regulator, prevents this excessive response by regulating GhMKK6 expression. ghr-miR5272a targets the GhMKK6 3′-untranslated region in cotton. Overexpressing miR5272a decreased the expression of GhMKK6 and disease-resistance genes, and increased sensitivity to F. oxysporum, yielding a similar phenotype to GhMKK6-silenced cotton. Overall, these results demonstrate that the ghr-miR5272a-mediated regulation of GhMKK6 expression contributes to the immune response in cotton, and reveal a new feedback loop mechanism in plant disease response.

BACKGROUND AND PURPOSE: Vascular inflammation, including the expression of inflammatory cytokines in endothelial cells, plays a critical role in hyperhomocysteinaemia‐associated vascular diseases. Cathepsin V, specifically expressed in humans, is involved in vascular diseases through its elastolytic and collagenolytic activities. The aim of this study was to determine the effects of cathepsin V on l‐homocysteine‐induced vascular inflammation. EXPERIMENTAL APPROACH: A high methionine diet‐induced hyperhomocysteinaemic mouse model was used to assess cathepsin V expression and vascular inflammation. Cultures of HUVECs were challenged with l‐homocysteine and the cathepsin L/V inhibitor SID to assess the pro‐inflammatory effects of cathepsin V. Transfection and antisense techniques were utilized to investigate the effects of cathepsin V on the dual‐specificity protein phosphatases (DUSPs) and MAPK pathways. KEY RESULTS: Cathepsin L (human cathepsin V homologous) was increased in the thoracic aorta endothelial cells of hyperhomocysteinaemic mice; l‐homocysteine promoted cathepsin V expression in HUVECs. SID suppressed the activity of cathepsin V and reversed the up‐regulation of inflammatory cytokines (IL‐6, IL‐8 and TNF‐α), adhesion and chemotaxis of leukocytes and vascular inflammation induced by l‐homocysteine in vivo and in vitro. Increased cathepsin V promoted the degradation of DUSP6 and DUSP7, phosphorylation and subsequent nuclear translocation of ERK1/2, phosphorylation of STAT1 and expression of IL‐6, IL‐8 and TNF‐α. CONCLUSIONS AND IMPLICATIONS: This study has identified a novel mechanism, which shows that l‐homocysteine‐induced upregulation of cathepsin V mediates vascular endothelial inflammation under high homocysteine condition partly via ERK(1/2)/STAT1 pathway. This mechanism could represent a potential therapeutic target in hyperaemia‐associated vascular diseases. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc

Using a systems-based approach, we have identified several genes not previously evaluated for a role(s) in chronological aging. Here, we have thoroughly investigated the chronological lifespan (CLS) of three of these genes (FUS3, KSS1 and HOG1) and their protein products, each of which have well-defined cell signaling roles in young cells. The importance of FUS3 and KSS1 in CLS are largely unknown and analyzed here for the first time. Using both qualitative and quantitative CLS assays, we show that deletion of any of the three MAPK's increases yeast lifespan. Furthermore, combined deletion of any MAPK and TOR1, most prominently fus3Δ/tor1Δ, produces a two-stage CLS response ending in lifespan increase greater than that of tor1Δ. Similar effects are achieved upon endogenous expression of a non-activatable form of Fus3. We speculate that the autophagy-promoting role of FUS3, which is inherently antagonistic to the role of TOR1, may in part be responsible for the differential aging phenotype of fus3Δ/tor1Δ. Consistent with this notion we show that nitrogen starvation, which promotes autophagy by deactivating Tor1, results in decreased CLS if FUS3 is deleted. Taken together, these results reveal a previously unrealized effect of mating-specific MAPKs in the chronological lifespan of yeast.

Isoacteoside (is a phenylethanoid isolated from Monochasma savatieri Franch. ex Maxim., which is an anti-inflammatory herb widely used in traditional Chinese medicine. However, the exact mechanism of the anti-inflammatory activity of isoacteoside is not completely understood. In this study, its anti-inflammatory mechanism was elucidated in mouse macrophages.The expression of the NF-κB pathway, MAPK pathway, iNOS, TNF-α, IL-6 and IL-1β was evaluated using Western blotting, quantitative real-time PCR or ELISA. TLR4 dimerization was determined by transfecting HEK293T cells with TLR4 plasmids. The in vivo anti-inflammatory effect of isoacteoside was determined using mouse models of xylene-induced ear oedema, LPS-induced endotoxic shock and LPS-induced endotoxaemia-associated acute kidney injury (AKI).Isoacteoside suppressed COX-2, iNOS, TNF-α, IL-6 and IL-1β expression. Furthermore, isoacteoside attenuated the LPS-induced transcriptional activity of NF-κB by decreasing the levels of phosphorylated IκB-α and IKK and NF-κB/p65 nuclear translocation. In addition, isoacteoside inhibited LPS-induced transcriptional activity of AP-1 by reducing the levels of phosphorylated JNK1/2 and p38MAPK. Isoacteoside blocked LPS-induced TLR4 dimerization, resulting in a reduction in the recruitment of MyD88 and TIR-domain-containing adapter-inducing interferon-β (TRIF) and the phosphorylation of TGF-β-activated kinase-1 (TAK1). Pretreatment of mice with isoacteoside effectively inhibited xylene-induced ear oedema and LPS-induced endotoxic death and protected against LPS-induced AKI.Isoacteoside blocked TLR4 dimerization, which activates the MyD88-TAK1-NF-κB/MAPK signalling cascades and TRIF pathway. Our data indicate that isoacteoside is a potential lead compound for the treatment of inflammatory diseases.

We evaluated the impact of premature cell death of antigen-presenting cells (APCs) by Caspase-1- and RipK3-signaling pathways on CD8+ T-cell priming during infection of mice with Salmonella typhimurium (ST). Our results indicate that Caspase1 and RipK3 synergize to rapidly eliminate infected APCs, which does not influence the initial activation of CD8+ T cells. However, the maintenance of primed CD8+ T cells was greatly compromised when both these pathways were disabled. Caspase-1- and RipK3-signaling did not influence NF-κB signaling in APCs, but synergized to promote processing of IL-1 and IL-18. Combined deficiency of Caspase1 and RipK3 resulted in compromised innate immunity and accelerated host fatality due to poor processing of IL-18. In contrast, synergism in cell death by Caspase-1- and RipK3 resulted in restriction of PD-1 and TIM3 expression on primed CD8+ T cells, which promoted the survival of activated CD8+ T cells.

Previously, we identified miR-125b as a key regulator of the undifferentiated state of hair follicle stem cells. Here, we show that in both mice and humans, miR-125b is abundantly expressed, particularly at early stages of malignant progression to squamous cell carcinoma (SCC), the second most prevalent cancer worldwide. Moreover, when elevated in normal murine epidermis, miR-125b promotes tumor initiation and contributes to malignant progression. We further show that miR-125b can confer “oncomiR addiction” in early stage malignant progenitors by delaying their differentiation and favoring an SCC cancer stem cell (CSC)-like transcriptional program. To understand how, we systematically identified and validated miR125b targets that are specifically associated with tumors that are dependent on miR-125b. Through molecular and genetic analysis of these targets, we uncovered new insights underlying miR-125b’s oncogenic function. Specifically, we show that, on the one hand, mir-125b directly represses stress-responsive MAP kinase genes and associated signaling. On the other hand, it indirectly prolongs activated (phosphorylated) EGFR signaling by repressing Vps4b (vacuolar protein-sorting 4 homolog B), encoding a protein implicated in negatively regulating the endosomal sorting complexes that are necessary for the recycling of active EGFR. Together, these findings illuminate miR-125b as an important microRNA regulator that is shared between normal skin progenitors and their early malignant counterparts.

Plants, which are sessile unlike most animals, have evolved a system to reduce growth under stress; however, the molecular mechanisms of this stress response are not well known. During programmed development, a fraction of the leaf epidermal precursor cells become meristemoid mother cells (MMCs), which are stem cells that produce both stomatal guard cells and epidermal pavement cells. Here we report that Arabidopsis plants, in response to osmotic stress, post-transcriptionally decrease the protein level of SPEECHLESS, the transcription factor promoting MMC identity, through the action of a mitogen-activated protein kinase (MAPK) cascade. The growth reduction under osmotic stress was lessened by inhibition of the MAPK cascade or by a mutation that disrupted the MAPK target amino acids in SPEECHLESS, indicating that Arabidopsis reduces growth under stress by integrating the osmotic stress signal into the MAPK-SPEECHLESS core developmental pathway.

Summary In this study it was found that ZFP36 is required for ABA-induced antioxidant defence and for the regulation of the cross-talk between NADPH oxidase, H2O2, and MAPK in ABA signalling., C2H2-type zinc finger proteins (ZFPs) have been shown to play important roles in the responses of plants to oxidative and abiotic stresses, and different members of this family might have different roles during stresses. Here a novel abscisic acid (ABA)- and hydrogen peroxide (H2O2)-responsive C2H2-type ZFP gene, ZFP36, is identified in rice. The analyses of ZFP36-overexpressing and silenced transgenic rice plants showed that ZFP36 is involved in ABA-induced up-regulation of the expression and the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). Overexpression of ZFP36 in rice plants was found to elevate the activities of antioxidant enzymes and to enhance the tolerance of rice plants to water stress and oxidative stress. In contrast, an RNA interference (RNAi) mutant of ZFP36 had lower activities of antioxidant enzymes and was more sensitive to water stress and oxidative stress. ABA-induced H2O2 production and ABA-activated mitogen-activated protein kinases (MAPKs) were shown to regulate the expression of ZFP36 in ABA signalling. On the other hand, ZFP36 also regulated the expression of NADPH oxidase genes, the production of H2O2, and the expression of OsMPK genes in ABA signalling. These results indicate that ZFP36 is required for ABA-induced antioxidant defence, for the tolerance of rice plants to water stress and oxidative stress, and for the regulation of the cross-talk between NADPH oxidase, H2O2, and MAPK in ABA signalling.

Summary A reduction in CO produced by IbHO occurs in leaves upon wounding and causes H2O2 generation and ERK phosphorylation. Defence systems are then switched on to protect plants from herbivores., Carbon monoxide (CO), one of the haem oxygenase (HO) products, plays important roles in plant development and stress adaptation. However, the function of CO involved in wounding responses is seldom studied. A wound-inducible gene, ipomoelin (IPO), of sweet potato (Ipomoea batatas cv. Tainung 57) was used as a target to study the regulation of CO in wounding responses. After wounding for 1h, the endogenous CO content and IbHO expression level were significantly reduced in leaves. IPO expression upon wounding was prohibited by the HO activator hemin, whereas the HO inhibitor zinc protoporphyrin IX elevated IPO expression. The IPO expression induced by wounding, H2O2, or methyl jasmonate was inhibited by CO. CO also affected the activities of ascorbate peroxidase, catalase, and peroxidase, and largely decreased H2O2 content in leaves. CO inhibited the extracellular signal-regulated kinase (ERK) phosphorylation induced by wounding. IbMAPK, the ERK of sweet potato, was identified by immunoblotting, and the interaction with its upstream activator, IbMEK1, was further confirmed by bimolecular fluorescence complementation and co-immunoprecipitation. Conclusively, wounding in leaves repressed IbHO expression and CO production, induced H2O2 generation and ERK phosphorylation, and then stimulated IPO expression.

Summary Excess red light induces a defence response by activating lipoxygenase at both the transcript and activity levels., Lipoxygenase (LOX), a non-haem-iron-containing dioxygenase, is activated under various biotic or abiotic stresses to trigger a series resistance response, but the molecular mechanism of LOX activation remains unclear. This work investigated the activation of LOX during the plant defence response induced by excess red light (RL). In conditions of RL-induced defence, Arabidopsis LOX activity and transcription levels of LOX2, LOX3, and LOX4 were both upregulated. Under RL, phytochrome B promoted the degradation of phytochrome-interacting factor 3 (PIF3), a factor that inhibited the expression levels of LOXs, and thus the transcription levels of LOX2, LOX3, and LOX4 were increased. Upon pathogen infection, the activity of mitogen-activated protein kinase 3 (MPK3) and MPK6 was increased in plants pre-treated with RL. Moreover, experiments with the inhibitor PD98059 and mutants mpk3 and mpk6-2 demonstrated that MPK3 and MPK6 were both responsible for LOX activation. Further results showed that, in response to RL, an increase in cytoplasmic calcium concentration and upregulation of calmodulin 3 (CaM3) transcript level occurred upstream of MPK3 and MPK6 activation. Collectively, these results suggested that activation of LOX both at the transcript level and in terms of activity modulates the defence response induced by RL, providing a new insight into the mechanistic study of LOX during plant defences.