Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP

DEFF Research Database (Denmark)

Peraldi, P; Frödin, M; Barnier, J V

1995-01-01

AMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21ras, but more important, this binding to p21ras is virtually...... abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP...

Autonomous and Non-autonomous Defects Underlie Hypertrophic Cardiomyopathy in BRAF-Mutant hiPSC-Derived Cardiomyocytes

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Rebecca Josowitz

2016-09-01

Full Text Available Germline mutations in BRAF cause cardio-facio-cutaneous syndrome (CFCS, whereby 40% of patients develop hypertrophic cardiomyopathy (HCM. As the role of the RAS/MAPK pathway in HCM pathogenesis is unclear, we generated a human induced pluripotent stem cell (hiPSC model for CFCS from three patients with activating BRAF mutations. By cell sorting for SIRPα and CD90, we generated a method to examine hiPSC-derived cell type-specific phenotypes and cellular interactions underpinning HCM. BRAF-mutant SIRPα+/CD90− cardiomyocytes displayed cellular hypertrophy, pro-hypertrophic gene expression, and intrinsic calcium-handling defects. BRAF-mutant SIRPα−/CD90+ cells, which were fibroblast-like, exhibited a pro-fibrotic phenotype and partially modulated cardiomyocyte hypertrophy through transforming growth factor β (TGFβ paracrine signaling. Inhibition of TGFβ or RAS/MAPK signaling rescued the hypertrophic phenotype. Thus, cell autonomous and non-autonomous defects underlie HCM due to BRAF mutations. TGFβ inhibition may be a useful therapeutic option for patients with HCM due to RASopathies or other etiologies.

Licochalcone A, a Polyphenol Present in Licorice, Suppresses UV-Induced COX-2 Expression by Targeting PI3K, MEK1, and B-Raf

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Nu Ry Song

2015-02-01

Full Text Available Licorice is a traditional botanical medicine, and has historically been commonly prescribed in Asia to treat various diseases. Glycyrrhizin (Gc, a triterpene compound, is the most abundant phytochemical constituent of licorice. However, high intake or long-term consumption of Gc has been associated with a number of side effects, including hypertension. However, the presence of alternative bioactive compounds in licorice with anti-carcinogenic effects has long been suspected. Licochalcone A (LicoA is a prominent member of the chalcone family and can be isolated from licorice root. To date, there have been no reported studies on the suppressive effect of LicoA against solar ultraviolet (sUV-induced cyclooxygenase (COX-2 expression and the potential molecular mechanisms involved. Here, we show that LicoA, a major chalcone compound of licorice, effectively inhibits sUV-induced COX-2 expression and prostaglandin E2 PGE2 generation through the inhibition of activator protein 1 AP-1 transcriptional activity, with an effect that is notably more potent than Gc. Western blotting analysis shows that LicoA suppresses sUV-induced phosphorylation of Akt/ mammalian target of rapamycin (mTOR and extracellular signal-regulated kinases (ERK1/2/p90 ribosomal protein S6 kinase (RSK in HaCaT cells. Moreover, LicoA directly suppresses the activity of phosphoinositide 3-kinase (PI3K, mitogen-activated protein kinase kinase (MEK1, and B-Raf, but not Raf-1 in cell-free assays, indicating that PI3K, MEK1, and B-Raf are direct molecular targets of LicoA. We also found that LicoA binds to PI3K and B-Raf in an ATP-competitive manner, although LicoA does not appear to compete with ATP for binding with MEK1. Collectively, these results provide insight into the biological action of LicoA, which may have potential for development as a skin cancer chemopreventive agent.

Towards a molecular classification of colorectal cancer: The role of BRAF

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Alexandra eThiel

2013-11-01

Full Text Available Different genetic aberrations of BRAF have been reported in various malignancies. BRAF is member of the RAS/RAF/MEK/ERK pathway and constitutive activity of this pathway can lead to increased cellular growth, invasion, and metastasis. The most common activating BRAF mutation in colorectal cancer is the V600E mutation, which is present in 5-15% of all tumors, and up to 80% of tumors with high microsatellite instability harbor this mutation. BRAF mutation is associated with proximal location, higher age, female gender, MSI-H, high grade, and mucinous histology, and is a marker of poor prognosis in colorectal cancer. The role of BRAF mutation as a predictive marker in respect of EGFR targeted treatments is controversial. BRAF V600 selective inhibitors have been approved for the treatment of V600 mutation positive metastatic melanoma, but the response rates in colorectal cancer are poor. This might be due to innate resistance mechanisms of colorectal cancers against the treatment solely targeting BRAF. To overcome resistance the combination of treatments, simultaneous inhibition of BRAF and MEK or PI3K/mTOR, might emerge as a successful therapeutic concept.

BRAF mutations in conjunctival melanoma

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Larsen, Ann-Cathrine; Dahl, Christina; Dahmcke, Christina M.

2016-01-01

with atypia. BRAF mutations were identified in 39 of 111 (35%) cases. The rate ratio of BRAF-mutated versus BRAF-wild-type melanoma did not change over time. BRAF mutations were associated with T1 stage (p = 0.007), young age (p = 0.001), male gender (p = 0.02), sun-exposed location (p = 0.01), mixed....../non-pigmented tumour colour (p = 0.02) and nevus origin (p = 0.005), but did not associate with prognosis. BRAF status in conjunctival melanoma and paired premalignant lesions corresponded in 19 of 20 cases. Immunohistochemistry detected BRAF V600E mutations with a sensitivity of 0.94 and a specificity of 1...

Combined BRAF and MEK Inhibition With Dabrafenib and Trametinib in BRAF V600–Mutant Colorectal Cancer

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Corcoran, Ryan B.; Atreya, Chloe E.; Falchook, Gerald S.; Kwak, Eunice L.; Ryan, David P.; Bendell, Johanna C.; Hamid, Omid; Messersmith, Wells A.; Daud, Adil; Kurzrock, Razelle; Pierobon, Mariaelena; Sun, Peng; Cunningham, Elizabeth; Little, Shonda; Orford, Keith; Motwani, Monica; Bai, Yuchen; Patel, Kiran; Venook, Alan P.; Kopetz, Scott

2015-01-01

Purpose To evaluate dabrafenib, a selective BRAF inhibitor, combined with trametinib, a selective MEK inhibitor, in patients with BRAF V600–mutant metastatic colorectal cancer (mCRC). Patients and Methods A total of 43 patients with BRAF V600–mutant mCRC were treated with dabrafenib (150 mg twice daily) plus trametinib (2 mg daily), 17 of whom were enrolled onto a pharmacodynamic cohort undergoing mandatory biopsies before and during treatment. Archival tissues were analyzed for microsatellite instability, PTEN status, and 487-gene sequencing. Patient-derived xenografts were established from core biopsy samples. Results Of 43 patients, five (12%) achieved a partial response or better, including one (2%) complete response, with duration of response > 36 months; 24 patients (56%) achieved stable disease as best confirmed response. Ten patients (23%) remained in the study > 6 months. All nine evaluable during-treatment biopsies had reduced levels of phosphorylated ERK relative to pretreatment biopsies (average decrease ± standard deviation, 47% ± 24%). Mutational analysis revealed that the patient achieving a complete response and two of three evaluable patients achieving a partial response had PIK3CA mutations. Neither PTEN loss nor microsatellite instability correlated with efficacy. Responses to dabrafenib plus trametinib were comparable in patient-derived xenograft–bearing mice and the biopsied lesions from each corresponding patient. Conclusion The combination of dabrafenib plus trametinib has activity in a subset of patients with BRAF V600–mutant mCRC. Mitogen-activated protein kinase signaling was inhibited in all patients evaluated, but to a lesser degree than observed in BRAF-mutant melanoma with dabrafenib alone. PIK3CA mutations were identified in responding patients and thus do not preclude response to this regimen. Additional studies targeting the mitogen-activated protein kinase pathway in this disease are warranted. PMID:26392102

Consistent absence of BRAF mutations in salivary gland carcinomas

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Nooshin Mohtasham

2017-06-01

Full Text Available Introduction: Malignant salivary gland tumors are rare entities. Despite advances in surgery, radiation therapy and chemotherapy, the rate of the mortality and five-year survival has not been improved markedly over the last few decades. The activation of EGFR- RAS-RAF signaling pathway contributes to the initiation and progression of many human cancers, promising a key pathway for therapeutic molecules. Thus, the objective of this study was to evaluate BRAF mutations in salivary gland carcinomas. Methods: We designed PCR- RFLP (Polymerase Chain Reaction -Restriction Fragment Length Polymorphism and screened 50 salivary gland carcinomas (SGCs including mucoepidermoid carcinoma (MEC, adenoid cystic carcinoma (AdCC and polymorphous low grade adenocarcinoma (PLGA for the BRAF V600E mutation. Results: PCR-RFLP analyses demonstrated no mutation in BRAF exon 15 for SGC samples at position V600, which is the most commonly mutated site for BRAF in human cancer. Conclusions: According to our results SGCs didn’t acquire BRAF mutations that result in a constitutive activation of the signaling cascade downstream of EGFR, hence SGCs can be a good candidate for anti EGFR therapies.

Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors

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Clare Judith Stones

2013-05-01

Full Text Available The NRAS and BRAF genes are frequently mutated in melanoma, suggesting that the NRAS-BRAF-MEK-ERK signalling pathway is an important target for therapy. Two classes of drugs, one targeting activated BRAF and one targeting MEK, are currently undergoing clinical evaluation. We have analysed the NRAS and BRAF mutational status of a series of 44 early passage lines developed from New Zealand patients with metastatic melanoma. 41% of the lines analysed had BRAF mutations, 23% had NRAS mutations and 36% had neither. We then determined IC50 values (drug concentrations for 50% growth inhibition for CI-1040, a commonly used inhibitor of MEK kinase; trametinib, a clinical agent targeting MEK kinase; and vemurafenib, an inhibitor of mutant BRAF kinase. Cell lines with activating BRAF mutations were significantly more sensitive to vemurafenib than lines with NRAS mutations or lines lacking either mutation (p < 0.001. IC50 values for CI-1040 and trametinib were strongly correlated (r = 0.98 with trametinib showing ~100-fold greater potency. Cell lines sensitive to vemurafenib were also sensitive to CI-1040 and trametinib, but there was no relationship between IC50 values and NRAS mutation status. A small number of lines lacking a BRAF mutation were sensitive to CI-1040 but resistant to vemurafenib. We used western blotting to investigate the effect on ERK phosphorylation of CI-1040 in four lines, of vemurafenib in two lines and of trametinib in two lines. The results support the view that MEK inhibitors might be combined with BRAF inhibitors in the treatment melanomas of with activated BRAF. The high sensitivity to trametinib of some lines with wild-type BRAF status also suggests that MEK inhibitors could have a therapeutic effect against some melanomas as single agents.

The "SWOT" of BRAF inhibition in melanoma: RAF inhibitors, MEK inhibitors or both?

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Nissan, Moriah H; Solit, David B

2011-12-01

Activating mutations in the BRAF gene are among the most prevalent kinase mutations in human cancer. BRAF mutations are most frequent in patients with melanoma where they occur in approximately 50% of patients with advanced disease. Remarkable clinical activity has recently been reported with highly selective RAF inhibitors in melanoma patients whose tumors harbor V600E BRAF mutations. The response rates of RAF inhibitors in patients with BRAF-mutant melanomas far exceed the activity level of any prior therapy studied in this disease. The results suggest that we have entered an era of personalized therapy for patients with metastatic melanoma in which treatment selection will be guided by BRAF mutational status. This review will discuss the strengths, weaknesses, opportunities and threats ("SWOT") of developing RAF and MEK selective inhibitors as anti-cancer therapies, recent insights into the mechanisms of intrinsic and acquired resistance to these agents, and current efforts to develop mechanism-based combination therapies.

Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1

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Meijia Yang

2017-09-01

Full Text Available BRAF-V600E (1799T > A is one of the most frequently reported driver mutations in multiple types of cancers, and patients with such mutations could benefit from selectively inactivating the mutant allele. Near this mutation site, there are two TTTN and one NGG protospacer-adjacent motifs (PAMs for Cpf1 and Cas9 CRISPR nucleases, respectively. The 1799T > A substitution also leads to the occurrence of a novel NGNG PAM for the EQR variant of Cas9. We examined the editing efficacy and selectivity of Cpf1, Cas9, and EQR variant to this mutation site. Only Cpf1 demonstrated robust activity to induce specific disruption of only mutant BRAF, not wild-type sequence. Cas9 recognized and cut both normal and mutant alleles, and no obvious gene editing events were observed using EQR variant. Our results support the potential applicability of Cpf1 in precision medicine through highly specific inactivation of many other gain-of-function mutations. Keywords: Cpf1, targeted therapy, BRAF V600E

The anti-apoptotic BAG3 protein is involved in BRAF inhibitor resistance in melanoma cells.

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Guerriero, Luana; Palmieri, Giuseppe; De Marco, Margot; Cossu, Antonio; Remondelli, Paolo; Capunzo, Mario; Turco, Maria Caterina; Rosati, Alessandra

2017-10-06

BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumour types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. In human melanomas, BAG3 positivity is correlated with the aggressiveness of the tumour cells and can sustain IKK-γ levels, allowing a sustained activation of NF-κB. Furthermore, BAG3 is able to modulate BRAFV600E levels and activity in thyroid carcinomas. BRAFV600E is the most frequent mutation detected in malignant melanomas and is targeted by Vemurafenib, a specific inhibitor found to be effective in the treatment of advanced melanoma. However, patients with BRAF-mutated melanoma may result insensitive ab initio or, mostly, develop acquired resistance to the treatment with this molecule. Here we show that BAG3 down-modulation interferes with BRAF levels in melanoma cells and sensitizes them to Vemurafenib treatment. Furthermore, the down-modulation of BAG3 protein in an in vitro model of acquired resistance to Vemurafenib can induce sensitization to the BRAFV600E specific inhibition by interfering with BRAF pathway through reduction of ERK phosphorylation, but also on parallel survival pathways. Future studies on BAG3 molecular interactions with key proteins responsible of acquired BRAF inhibitor resistance may represent a promising field for novel multi-drugs treatment design.

Degree of Tissue Differentiation Dictates Susceptibility to BRAF-Driven Colorectal Cancer

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Kevin Tong

2017-12-01

Full Text Available Oncogenic mutations in BRAF are believed to initiate serrated colorectal cancers; however, the mechanisms of BRAF-driven colon cancer are unclear. We find that oncogenic BRAF paradoxically suppresses stem cell renewal and instead promotes differentiation. Correspondingly, tumor formation is inefficient in BRAF-driven mouse models of colon cancer. By reducing levels of differentiation via genetic manipulation of either of two distinct differentiation-promoting factors (Smad4 or Cdx2, stem cell activity is restored in BRAFV600E intestines, and the oncogenic capacity of BRAFV600E is amplified. In human patients, we observe that reduced levels of differentiation in normal tissue is associated with increased susceptibility to serrated colon tumors. Together, these findings help resolve the conditions necessary for BRAF-driven colon cancer initiation. Additionally, our results predict that genetic and/or environmental factors that reduce tissue differentiation will increase susceptibility to serrated colon cancer. These findings offer an opportunity to identify susceptible individuals by assessing their tissue-differentiation status.

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS.

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Zuidervaart, W; van Nieuwpoort, F; Stark, M; Dijkman, R; Packer, L; Borgstein, A-M; Pavey, S; van der Velden, P; Out, C; Jager, M J; Hayward, N K; Gruis, N A

2005-06-06

In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma.

Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors.

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Bijay S Jaiswal

Full Text Available BACKGROUND: Oncogenic RAS is a highly validated cancer target. Attempts at targeting RAS directly have so far not succeeded in the clinic. Understanding downstream RAS-effectors that mediate oncogenesis in a RAS mutant setting will help tailor treatments that use RAS-effector inhibitors either alone or in combination to target RAS-driven tumors. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have investigated the sufficiency of targeting RAS-effectors, RAF, MEK and PI3-Kinase either alone or in combination in RAS mutant lines, using an inducible shRNA in vivo mouse model system. We find that in colon cancer cells harboring a KRAS(G13D mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo. In melanoma cells harboring an NRAS(Q61L or NRAS(Q61K mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy. CONCLUSION/SIGNIFICANCE: Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy. This can be achieved by either targeting both BRAF and CRAF or BRAF and PIK3CA simultaneously in NRAS mutant tumor cells.

B-Raf and CRHR1 internalization mediate biphasic ERK1/2 activation by CRH in hippocampal HT22 Cells.

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Bonfiglio, Juan J; Inda, Carolina; Senin, Sergio; Maccarrone, Giuseppina; Refojo, Damián; Giacomini, Damiana; Turck, Christoph W; Holsboer, Florian; Arzt, Eduardo; Silberstein, Susana

2013-03-01

CRH is a key regulator of neuroendocrine, autonomic, and behavioral response to stress. CRH-stimulated CRH receptor 1 (CRHR1) activates ERK1/2 depending on intracellular context. In a previous work, we demonstrated that CRH activates ERK1/2 in limbic areas of the mouse brain (hippocampus and basolateral amygdala). ERK1/2 is an essential mediator of hippocampal physiological processes including emotional behavior, synaptic plasticity, learning, and memory. To elucidate the molecular mechanisms by which CRH activates ERK1/2 in hippocampal neurons, we used the mouse hippocampal cell line HT22. We document for the first time that ERK1/2 activation in response to CRH is biphasic, involving a first cAMP- and B-Raf-dependent early phase and a second phase that critically depends on CRHR1 internalization and β-arrestin2. By means of mass-spectrometry-based screening, we identified B-Raf-associated proteins that coimmunoprecipitate with endogenous B-Raf after CRHR1 activation. Using molecular and pharmacological tools, the functional impact of selected B-Raf partners in CRH-dependent ERK1/2 activation was dissected. These results indicate that 14-3-3 proteins, protein kinase A, and Rap1, are essential for early CRH-induced ERK1/2 activation, whereas dynamin and vimentin are required for the CRHR1 internalization-dependent phase. Both phases of ERK1/2 activation depend on calcium influx and are affected by calcium/calmodulin-dependent protein kinase II inactivation. Thus, this report describes the dynamics and biphasic nature of ERK1/2 activation downstream neuronal CRHR1 and identifies several new critical components of the CRHR1 signaling machinery that selectively controls the early and late phases of ERK1/2 activation, thus providing new potential therapeutic targets for stress-related disorders.

Clinical, pathologic, and biologic features associated with BRAF mutations in non-small cell lung cancer.

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Cardarella, Stephanie; Ogino, Atsuko; Nishino, Mizuki; Butaney, Mohit; Shen, Jeanne; Lydon, Christine; Yeap, Beow Y; Sholl, Lynette M; Johnson, Bruce E; Jänne, Pasi A

2013-08-15

BRAF mutations are found in a subset of non-small cell lung cancers (NSCLC). We examined the clinical characteristics and treatment outcomes of patients with NSCLC harboring BRAF mutations. Using DNA sequencing, we successfully screened 883 patients with NSCLC for BRAF mutations between July 1, 2009 and July 16, 2012. Baseline characteristics and treatment outcomes were compared between patients with and without BRAF mutations. Wild-type controls consisted of patients with NSCLC without a somatic alteration in BRAF, KRAS, EGFR, and ALK. In vitro studies assessed the biologic properties of selected non-V600E BRAF mutations identified from patients with NSCLC. Of 883 tumors screened, 36 (4%) harbored BRAF mutations (V600E, 18; non-V600E, 18) and 257 were wild-type for BRAF, EGFR, KRAS, and ALK negative. Twenty-nine of 36 patients with BRAF mutations were smokers. There were no distinguishing clinical features between BRAF-mutant and wild-type patients. Patients with advanced NSCLC with BRAF mutations and wild-type tumors showed similar response rates and progression-free survival (PFS) to platinum-based combination chemotherapy and no difference in overall survival. Within the BRAF cohort, patients with V600E-mutated tumors had a shorter PFS to platinum-based chemotherapy compared with those with non-V600E mutations, although this did not reach statistical significance (4.1 vs. 8.9 months; P = 0.297). We identified five BRAF mutations not previously reported in NSCLC; two of five were associated with increased BRAF kinase activity. BRAF mutations occur in 4% of NSCLCs and half are non-V600E. Prospective trials are ongoing to validate BRAF as a therapeutic target in NSCLC. ©2013 AACR.

Phase II trial (BREAK-2) of the BRAF inhibitor dabrafenib (GSK2118436) in patients with metastatic melanoma.

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Ascierto, Paolo A; Minor, David; Ribas, Antoni; Lebbe, Celeste; O'Hagan, Anne; Arya, Niki; Guckert, Mary; Schadendorf, Dirk; Kefford, Richard F; Grob, Jean-Jacques; Hamid, Omid; Amaravadi, Ravi; Simeone, Ester; Wilhelm, Tabea; Kim, Kevin B; Long, Georgina V; Martin, Anne-Marie; Mazumdar, Jolly; Goodman, Vicki L; Trefzer, Uwe

2013-09-10

Dabrafenib (GSK2118436) is a potent inhibitor of mutated BRAF kinase. Our multicenter, single-arm, phase II study assessed the safety and clinical activity of dabrafenib in BRAF(V600E/K) mutation-positive metastatic melanoma (mut(+) MM). Histologically confirmed patients with stage IV BRAF(V600E/K) mut(+) MM received oral dabrafenib 150 mg twice daily until disease progression, death, or unacceptable adverse events (AEs). The primary end point was investigator-assessed overall response rate in BRAF(V600E) mut(+) MM patients. Secondary end points included progression-free survival (PFS) and overall survival (OS). Exploratory objectives included the comparison of BRAF mutation status between tumor-specific circulating cell-free DNA (cfDNA) and tumor tissue, and the evaluation of cfDNA as a predictor of clinical outcome. Seventy-six patients with BRAF(V600E) and 16 patients with BRAF(V600K) mut(+) MM were enrolled onto the study. In the BRAF(V600E) group, 45 patients (59%) had a confirmed response (95% CI, 48.2 to 70.3), including five patients (7%) with complete responses. Two patients (13%) with BRAF(V600K) mut(+) MM had a confirmed partial response (95% CI, 0 to 28.7). In the BRAF(V600E) and BRAF(V600K) groups, median PFS was 6.3 months and 4.5 months, and median OS was 13.1 months and 12.9 months, respectively. The most common AEs were arthralgia (33%), hyperkeratosis (27%), and pyrexia (24%). Overall, 25 patients (27%) experienced a serious AE and nine patients (10%) had squamous cell carcinoma. Baseline cfDNA levels predicted response rate and PFS in BRAF(V600E) mut(+) MM patients. Dabrafenib was well tolerated and clinically active in patients with BRAF(V600E/K) mut(+) MM. cfDNA may be a useful prognostic and response marker in future studies.

Small molecules against B-RAF (BRAF Val600Glu (V600E single mutation

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Zaharie F

2015-07-01

Full Text Available Florin Zaharie,1,* Roxana Cojocneanu-Petric,1,* Mihai Muresan,1 Ioana Frinc,2 Delia Dima,2 Bobe Petrushev,3 Alina Tanase,4 Cristian Berce,1 Mariana Chitic,2 Ioana Berindan-Neagoe,1 Valentina Pileczki,1 Alexandru Irimie,5 Ciprian Tomuleasa2 1Iuliu Hatieganu University of Medicine and Pharmacy, 2Department of Hematology, Ion Chiricuta Oncology Institute, 3Department of Pathology, Emergency University Hospital, Cluj Napoca, 4Department of Stem Cell Transplantation, Fundeni Clinical Institute, Bucharest, 5Department of Surgery, Ion Chiricuta Oncology Institute, Cluj Napoca, Romania *These authors contributed equally to this communicationWe have read with great interest the paper by Tang and Chen1 published in the most recent issue of the International Journal of Nanomedicine, in which the authors describe the protocol by which scientists constructed the ideal BRAF (V600E-modeled structure through homology modeling and introduced the method of structure-based docking or virtual screening from a large compound database. They concluded that BRAF (V600E has a quite prominent structural or conformational variation when compared to the wild-type BRAF protein by matrix of root mean square fluctuation and principal component analysis. On the basis of structure-based virtual screening, ligand-based quantitative structure activity relationship models, and molecular dynamics simulation, we recommend aknadicine and 16beta-hydroxy-19s-vindolinine N-oxide as potent compounds for developing novel inhibitors in the future. Read the original article 

BRAF-V600E mutant papillary craniopharyngioma dramatically responds to combination BRAF and MEK inhibitors.

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Roque, Ashley; Odia, Yazmin

2017-04-01

We present a patient with BRAF-V600E mutant papillary craniopharyngioma successfully treated with combination BRAF (dabrafenib 150 mg twice daily) and MEK (trametinib 2 mg daily) inhibitors after her unresectable tumor proved refractory to radiation. Serial brain MRIs and PET revealed marked tumor reduction with gradual neurological improvement and permanent panhypopituitarism.

Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells.

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Mologni, Luca; Costanza, Mariantonia; Sharma, Geeta Geeta; Viltadi, Michela; Massimino, Luca; Citterio, Stefania; Purgante, Stefania; Raman, Hima; Pirola, Alessandra; Zucchetti, Massimo; Piazza, Rocco; Gambacorti-Passerini, Carlo

2018-05-01

BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAF V600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion within an amplified BRAF V600E locus and a missense point mutation of the transcriptional repressor BCORL1 in vemurafenib-resistant A375 melanoma cells. Functional data confirmed that truncated p47BRAF V600E and mutant BCORL1 Q1076H both contribute to resistance. Interestingly, either endogenous BCORL1 silencing or ectopic BCORL1 Q1076H expression mimicked the effects of a CRISPR/Cas9-edited BCORL1 Q1076H locus, suggesting a complex mixture of loss- and gain-of-function effects caused by the mutation. Transcriptomic data confirmed this hypothesis. Finally, we show that the pan-RAF inhibitor sorafenib is not affected by expression of BRAF deletion variant and effectively synergizes with vemurafenib to block resistant cells, suggesting a possible intervention for this class of mutants. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Intercellular Resistance to BRAF Inhibition Can Be Mediated by Extracellular Vesicle–Associated PDGFRβ

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Laura J. Vella

2017-11-01

Full Text Available Treatment of BRAF mutant melanoma with kinase inhibitors has been associated with rapid tumor regression; however, this clinical benefit is short-lived, and most patients relapse. A number of studies suggest that the extracellular environment promotes BRAF inhibitor resistance and tumor progression. Extracellular vesicles, such as exosomes, are functional mediators in the extracellular environment. They are small vesicles known to carry a concentrated group of functional cargo and serve as intercellular communicators not only locally but also systemically. Increasingly, it is reported that extracellular vesicles facilitate the development of drug resistance in cancer; however, their role in BRAF inhibitor resistance in melanoma is unclear. Here we investigated if extracellular vesicles from BRAF inhibitor–resistant melanoma could influence drug sensitivity in recipient melanoma cells. We demonstrate that the resistance driver, PDGFRβ, can be transferred to recipient melanoma cells via extracellular vesicles, resulting in a dose-dependent activation of PI3K/AKT signaling and escape from MAPK pathway BRAF inhibition. These data suggest that the BRAF inhibitor–sensitive phenotype of metastatic melanoma can be altered by delivery of PDGFRβ by extracellular vesicles derived from neighboring drug-resistant melanoma cells.

BRAF mutation testing in solid tumors: a methodological comparison.

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Weyant, Grace W; Wisotzkey, Jeffrey D; Benko, Floyd A; Donaldson, Keri J

2014-09-01

Solid tumor genotyping has become standard of care for the characterization of proto-oncogene mutational status, which has traditionally been accomplished with Sanger sequencing. However, companion diagnostic assays and comparable laboratory-developed tests are becoming increasingly popular, such as the cobas 4800 BRAF V600 Mutation Test and the INFINITI KRAS-BRAF assay, respectively. This study evaluates and validates the analytical performance of the INFINITI KRAS-BRAF assay and compares concordance of BRAF status with two reference assays, the cobas test and Sanger sequencing. DNA extraction from FFPE tissue specimens was performed followed by multiplex PCR amplification and fluorescent label incorporation using allele-specific primer extension. Hybridization to a microarray, signal detection, and analysis were then performed. The limits of detection were determined by testing dilutions of mutant BRAF alleles within wild-type background DNA, and accuracy was calculated based on these results. The INFINITI KRAS-BRAF assay produced 100% concordance with the cobas test and Sanger sequencing and had sensitivity equivalent to the cobas assay. The INFINITI assay is repeatable with at least 95% accuracy in the detection of mutant and wild-type BRAF alleles. These results confirm that the INFINITI KRAS-BRAF assay is comparable to traditional sequencing and the Food and Drug Administration-approved companion diagnostic assay for the detection of BRAF mutations. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Forebrain-specific knockout of B-raf kinase leads to deficits in hippocampal long-term potentiation, learning, and memory.

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Chen, Adele P; Ohno, Masuo; Giese, K Peter; Kühn, Ralf; Chen, Rachel L; Silva, Alcino J

2006-01-01

Raf kinases are downstream effectors of Ras and upstream activators of the MEK-ERK cascade. Ras and MEK-ERK signaling play roles in learning and memory (L&M) and neural plasticity, but the roles of Raf kinases in L&M and plasticity are unclear. Among Raf isoforms, B-raf is preferentially expressed in the brain. To determine whether B-raf has a role in synaptic plasticity and L&M, we used the Cre-LoxP gene targeting system to derive forebrain excitatory neuron B-raf knockout mice. This conditional knockout resulted in deficits in ERK activation and hippocampal long-term potentiation (LTP) and impairments in hippocampus-dependent L&M, including spatial learning and contextual discrimination. Despite the widespread expression of B-raf, this mutation did not disrupt other forms of L&M, such as cued fear conditioning and conditioned taste aversion. Our findings demonstrate that B-raf plays a role in hippocampal ERK activation, synaptic plasticity, and L&M.

Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells

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Luca Mologni; Mariantonia Costanza; Geeta Geeta Sharma; Michela Viltadi; Luca Massimino; Stefania Citterio; Stefania Purgante; Hima Raman; Alessandra Pirola; Massimo Zucchetti; Rocco Piazza; Carlo Gambacorti-Passerini

2018-01-01

BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAFV600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion w...

Efficacy of sorafenib in BRAF-mutated non-small-cell lung cancer (NSCLC) and no response in synchronous BRAF wild type-hepatocellular carcinoma: a case report

International Nuclear Information System (INIS)

Casadei Gardini, Andrea; Chiadini, Elisa; Faloppi, Luca; Marisi, Giorgia; Delmonte, Angelo; Scartozzi, Mario; Loretelli, Cristian; Lucchesi, Alessandro; Oboldi, Devil; Dubini, Alessandra; Frassineti, Giovanni Luca; Ulivi, Paola

2016-01-01

Sorafenib is a multi-targeted kinase inhibitor with a demonstrated activity in renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC), and it is currently used for the treatment of these pathologies. Ongoing clinical trials are studying its activity in other malignancies, such as non-small-cell lung cancer (NSCLC). However, no biological marker is known to define either the sensitivity or resistance to the drug. Here we report a case of a patient with two synchronous tumors, HCC and NSCLC, with metastases in the contralateral lung and bone. The patient was treated with gemcitabine as first line, with a resulting progressive disease after two months, and then with sorafenib at standard dosage in the second line setting. After 6 months of treatment CT scan showed a partial response in the primary lesion of the lung, complete response of the metastasis in the contralateral lung, and stability of HCC. The patient had progression in the lung, liver and bone after 13 months of therapy. A molecular characterization of NSCLC and HCC lesions was performed, revealing a BRAF exon 11 mutation (G469V) only in NSCLC. We hypothesize that the response observed in NSCLC lesions could be due to the presence of BRAF mutation, and that this alteration could be responsible in determining sorafenib sensitivity. Results observed in this case encourage further research on the activity of sorafenib in both HCC and NSCLC, based on the presence of BRAF mutation. This could lead to a selection of HCC patients to be treated with this drug, and could help identify a novel treatment strategy for BRAF-mutated NSCLC patients

Strategies for Overcoming Resistance in Tumours Harboring BRAF Mutations

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Nourah Mohammad Obaid

2017-03-01

Full Text Available The development of resistance to previously effective treatments has been a challenge for health care providers and a fear for patients undergoing cancer therapy. This is an unfortunately frequent occurrence for patients undergoing targeted therapy for tumours harboring the activating V600E mutation of the BRAF gene. Since the initial identification of the BRAF mutation in 2002, a series of small molecular inhibitors that target the BRAFV600E have been developed, but intrinsic and acquired resistance to these drugs has presented an ongoing challenge. More recently, improvements in therapy have been achieved by combining the use of BRAF inhibitors with other drugs, such as inhibitors of the downstream effector mitogen activated protein kinase (MAPK/extracellular-signal regulated kinase (ERK kinase (MEK. Despite improved success in response rates and in delaying resistance using combination therapy, ultimately, the acquisition of resistance remains a concern. Recent research articles have shed light on some of the underlying mechanisms of this resistance and have proposed numerous strategies that might be employed to overcome or avoid resistance to targeted therapies. This review will explore some of the resistance mechanisms, compare what is known in melanoma cancer to colorectal cancer, and discuss strategies under development to manage the development of resistance.

Synthesis, biological evaluation and molecular docking of novel 5-phenyl-1H-pyrazol derivatives as potential BRAF(V600E) inhibitors.

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Dong, Jing-Jun; Li, Qing-Shan; Wang, Shu-Fu; Li, Cui-Yun; Zhao, Xin; Qiu, Han-Yue; Zhao, Meng-Yue; Zhu, Hai-Liang

2013-10-07

The RAF-MEK-ERK cascade appears to be intimately involved in the regulation of cell cycle progression and apoptosis. The BRAF(V600E) mutant results in constitutive activation of the ERK pathway, which can lead to cellular growth dysregulation. A series of 5-phenyl-1H-pyrazol derivatives (3a-5e) have been designed and synthesized, and their biological activities were evaluated as potential BRAF(V600E) inhibitors. All the compounds were reported for the first time except 3e, and compound 1-(4-bromo-2-hydroxybenzyl)-3-phenyl-1-(5-phenyl-1H-pyrazol-3-yl)urea (5c) displayed the most potent inhibitory activity (BRAF(V600E) IC50 = 0.19 μM). Antiproliferative assay results indicated that compound 5c possessed high antiproliferative activity against cell lines WM266.4 and A375 in vitro, with IC50 values of 1.50 and 1.32 μM, respectively, which were comparable with the positive control vemurafenib. Docking simulations showed that compound 5c binds tightly to the BRAF(V600E) active site and acts as BRAF(V600E) inhibitor. A 3D-QSAR model was also built to provide more pharmacophore understanding towards designing new agents with more potent BRAF(V600E) inhibitory activity.

Docking based 3d-QSAR studies applied at the BRAF inhibitors to understand the binding mechanism

International Nuclear Information System (INIS)

Mahmood, U.; Haq, Z.U.

2011-01-01

BRAF is a great therapeutic target in a wide variety of human cancers. It is the member of Ras Activating Factor (RAF) family of serine/throenine kinase. The mutated form of the BRAF has diverted all the attention towards itself because of increase severity and elevated kinase activity. The RAF signal transduction cascade is a conserved protein pathway that is involved in cell cycle progression and apoptosis. The ERK regulates phosphorylation of different proteins either in cytosol or in nucleus but disorders in ERK signaling pathway cause mutation in BRAF. This cascade in these cells may provide selection of mutated BRAF in which valine is substituted with glutamatic acid at position 600. This mutation occurs in activation loop. A number of inhibitors reported to target different members of RAF, some of them have potential to target the BRAF as well. Major reason for failure of previously reported inhibitors was due to the highly conserved sequence and confirmation of catalytic cleft which is always a center of consideration for binding of inhibitors to suppress the kinase activity. This is the first attempt to study and understand the BARF inhibitors - protein interactions in detail by utilizing 3D-QSAR and molecular docking techniques. Most reliable techniques of 3D QSAR i.e Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) were applied for three different data sets. The data sets selected for better evaluation of BRAF inhibitors belongs to 2, 6-Disubstituted Pyrazine, Pyridoimidazolones and its derivatives. Our models would offer help to better understand the structure-activity relationships that exist for these classes of compounds and also facilitate the design of novel inhibitors with good chemical diversity. (Author)

Sustained ERK inhibition maximizes responses of BrafV600E thyroid cancers to radioiodine.

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Nagarajah, James; Le, Mina; Knauf, Jeffrey A; Ferrandino, Giuseppe; Montero-Conde, Cristina; Pillarsetty, Nagavarakishore; Bolaender, Alexander; Irwin, Christopher; Krishnamoorthy, Gnana Prakasam; Saqcena, Mahesh; Larson, Steven M; Ho, Alan L; Seshan, Venkatraman; Ishii, Nobuya; Carrasco, Nancy; Rosen, Neal; Weber, Wolfgang A; Fagin, James A

2016-11-01

Radioiodide (RAI) therapy of thyroid cancer exploits the relatively selective ability of thyroid cells to transport and accumulate iodide. Iodide uptake requires expression of critical genes that are involved in various steps of thyroid hormone biosynthesis. ERK signaling, which is markedly increased in thyroid cancer cells driven by oncogenic BRAF, represses the genetic program that enables iodide transport. Here, we determined that a critical threshold for inhibition of MAPK signaling is required to optimally restore expression of thyroid differentiation genes in thyroid cells and in mice with BrafV600E-induced thyroid cancer. Although the MEK inhibitor selumetinib transiently inhibited ERK signaling, which subsequently rebounded, the MEK inhibitor CKI suppressed ERK signaling in a sustained manner by preventing RAF reactivation. A small increase in ERK inhibition markedly increased the expression of thyroid differentiation genes, increased iodide accumulation in cancer cells, and thereby improved responses to RAI therapy. Only a short exposure to the drug was necessary to obtain a maximal response to RAI. These data suggest that potent inhibition of ERK signaling is required to adequately induce iodide uptake and indicate that this is a promising strategy for the treatment of BRAF-mutant thyroid cancer.

BRAF Gene Copy Number and Mutant Allele Frequency Correlate with Time to Progression in Metastatic Melanoma Patients Treated with MAPK Inhibitors.

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Stagni, Camilla; Zamuner, Carolina; Elefanti, Lisa; Zanin, Tiziana; Bianco, Paola Del; Sommariva, Antonio; Fabozzi, Alessio; Pigozzo, Jacopo; Mocellin, Simone; Montesco, Maria Cristina; Chiarion-Sileni, Vanna; De Nicolo, Arcangela; Menin, Chiara

2018-06-01

Metastatic melanoma is characterized by complex genomic alterations, including a high rate of mutations in driver genes and widespread deletions and amplifications encompassing various chromosome regions. Among them, chromosome 7 is frequently gained in BRAF -mutant melanoma, inducing a mutant allele-specific imbalance. Although BRAF amplification is a known mechanism of acquired resistance to therapy with MAPK inhibitors, it is still unclear if BRAF copy-number variation and BRAF mutant allele imbalance at baseline can be associated with response to treatment. In this study, we used a multimodal approach to assess BRAF copy number and mutant allele frequency in pretreatment melanoma samples from 46 patients who received MAPK inhibitor-based therapy, and we analyzed the association with progression-free survival. We found that 65% patients displayed BRAF gains, often supported by chromosome 7 polysomy. In addition, we observed that 64% patients had a balanced BRAF -mutant/wild-type allele ratio, whereas 14% and 23% patients had low and high BRAF mutant allele frequency, respectively. Notably, a significantly higher risk of progression was observed in patients with a diploid BRAF status versus those with BRAF gains [HR, 2.86; 95% confidence interval (CI), 1.29-6.35; P = 0.01] and in patients with low percentage versus those with a balanced BRAF mutant allele percentage (HR, 4.54; 95% CI, 1.33-15.53; P = 0.016). Our data suggest that quantitative analysis of the BRAF gene could be useful to select the melanoma patients who are most likely to benefit from therapy with MAPK inhibitors. Mol Cancer Ther; 17(6); 1332-40. ©2018 AACR . ©2018 American Association for Cancer Research.

Design and discovery of thioether and nicotinamide containing sorafenib analogues as multikinase inhibitors targeting B-Raf, B-RafV600E and VEGFR-2.

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Sun, Shaofeng; He, Zuopeng; Huang, Mindong; Wang, Ningning; He, Zongzhong; Kong, Xiangkai; Yao, Jianwen

2018-04-03

New sorafenib derivatives containing thioether and nicotinamide moiety were designed and synthesized as B-Raf, B-Raf V600E and VEGFR-2 multikinase inhibitors. Their in vitro enzymatic inhibitory activities against B-Raf, B-Raf V600E and VEGFR-2 and their antiproliferative activities against HCT-116 and B16BL6 cell lines were evaluated and described. Most of the compounds showed potent activities against both cell lines and specific kinases. Compounds a1, b1 and c4, which exhibited the most potent inhibitory activities against B-Raf with IC 50 of 21 nM, 27 nM and 17 nM, B-Raf V600E with IC 50 of 29 nM, 28 nM and 16 nM, VEGFR-2 with IC 50 of 84 nM, 46 nM and 63 nM, respectively, and good antiproliferative activities, also demonstrated competitive antiangiogenic activities to sorafenib in in vitro HUVEC tube formation assay. Copyright © 2018 Elsevier Ltd. All rights reserved.

Mutação BRAF em pacientes idosos submetidos à tireoidectomia BRAF mutation in the elderly submitted to thyroidectomy

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Antonio Augusto T. Bertelli

2013-04-01

Full Text Available OBJETIVO: Avaliar a frequência da mutação V600E do gene BRAF em pacientes com mais de 65 anos de idade submetidos à tireoidectomia, correlacionando sua presença ou ausência com as diferentes lesões histológicas, com as variantes e com fatores prognósticos do carcinoma papilífero. MÉTODOS: Foram avaliados 85 pacientes com mais de 65 anos de idade submetidos à tireoidectomia, analisando a mutação BRAF V600E através de reação de PCR-RT realizada após a extração do DNA dos blocos de parafina. RESULTADOS: Detectou-se ausência ou presença da mutação BRAF V600E em 47 pacientes (55,3%. Entre os 17 carcinomas papilíferos estudados, sete apresentavam a mutação (41,2%. Demonstrou-se associação estatística entre a presença desta mutação e a variante clássica do carcinoma papilífero, além de tendência de associação com o extravasamento tireoideano. CONCLUSÃO: A mutação BRAF nos pacientes idosos também é exclusiva do carcinoma papilífero e tem frequência expressiva. Além disso, está relacionada à variante clássica e, possivelmente, ao extravasamento tireoideano.OBJECTIVE: To evaluate the frequency of the BRAF V600E mutation in patients over 65 years of age undergoing thyroidectomy, correlating its presence or absence with the different histologic lesions, their variants and with prognostic factors of papillary carcinoma. METHODS: We evaluated 85 patients over 65 years of age who underwent thyroidectomy, analyzing the BRAF V600E mutation by RT-PCR performed after DNA extraction from the paraffin blocks. RESULTS: The study detected the presence or absence of BRAF V600E mutation in 47 patients (55.3%. Among the 17 papillary carcinomas studied, seven had the mutation (41.2%. There was a statistical association between the presence of this mutation and the classic variant of papillary carcinoma, and a trend of association with thyroid extravasation. CONCLUSION: BRAF mutation in the elderly is also exclusive of

BRAF-mutant melanoma: treatment approaches, resistance mechanisms, and diagnostic strategies

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Spagnolo F

2015-01-01

Full Text Available Francesco Spagnolo,1 Paola Ghiorzo,2,3 Laura Orgiano,4 Lorenza Pastorino,2,3 Virginia Picasso,4 Elena Tornari,4 Vincenzo Ottaviano,4 Paola Queirolo4 1Department of Plastic and Reconstructive Surgery, IRCCS Azienda Ospedaliera Universitaria San Martino, IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; 2Department of Internal Medicine and Medical Specialties (DiMI, University of Genoa, Genova, Italy; 3Genetics of Rare Cancers, IRCCS Azienda Ospedaliera Universitaria San Martino, IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy; 4Department of Medical Oncology, IRCCS Azienda Ospedaliera Universitaria San Martino, IST Istituto Nazionale per la Ricerca sul Cancro, Genova, Italy Abstract: BRAF inhibitors vemurafenib and dabrafenib achieved improved overall survival over chemotherapy and have been approved for the treatment of BRAF-mutated metastatic melanoma. More recently, the combination of BRAF inhibitor dabrafenib with MEK inhibitor trametinib has shown improved progression-free survival, compared to dabrafenib monotherapy, in a Phase II study and has received approval by the US Food and Drug Administration. However, even when treated with the combination, most patients develop mechanisms of acquired resistance, and some of them do not achieve tumor regression at all, because of intrinsic resistance to therapy. Along with the development of BRAF inhibitors, immunotherapy made an important step forward: ipilimumab, an anti-CTLA-4 monoclonal antibody, was approved for the treatment of metastatic melanoma; anti-PD-1 agents achieved promising results in Phase I/II trials, and data from Phase III studies will be ready soon. The availability of such drugs, which are effective regardless of BRAF status, has made the therapeutic approach more complex, as first-line treatment with BRAF inhibitors may not be the best choice for all BRAF-mutated patients. The aim of this paper is to review the systemic therapeutic options

An Investigation of Molecular Docking and Molecular Dynamic Simulation on Imidazopyridines as B-Raf Kinase Inhibitors

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Huiding Xie

2015-11-01

Full Text Available In the recent cancer treatment, B-Raf kinase is one of key targets. Nowadays, a group of imidazopyridines as B-Raf kinase inhibitors have been reported. In order to investigate the interaction between this group of inhibitors and B-Raf kinase, molecular docking, molecular dynamic (MD simulation and binding free energy (ΔGbind calculation were performed in this work. Molecular docking was carried out to identify the key residues in the binding site, and MD simulations were performed to determine the detail binding mode. The results obtained from MD simulation reveal that the binding site is stable during the MD simulations, and some hydrogen bonds (H-bonds in MD simulations are different from H-bonds in the docking mode. Based on the obtained MD trajectories, ΔGbind was computed by using Molecular Mechanics Generalized Born Surface Area (MM-GBSA, and the obtained energies are consistent with the activities. An energetic analysis reveals that both electrostatic and van der Waals contributions are important to ΔGbind, and the unfavorable polar solvation contribution results in the instability of the inhibitor with the lowest activity. These results are expected to understand the binding between B-Raf and imidazopyridines and provide some useful information to design potential B-Raf inhibitors.

BRAF V600E mutation in papillary thyroid cancer and its effect on postoperative radioiodine (131I) therapy: Should we modify our therapeutic strategy?

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Domínguez Ayala, Maite; Expósito Rodríguez, Amaia; Bilbao González, Amaia; Mínguez Gabiña, Pablo; Gutiérrez Rodríguez, Teresa; Rodeño Ortiz de Zarate, Emilia; García Carrillo, Maitane; Barrios Treviño, Borja

2018-03-19

The BRAF V600E mutation in papillary thyroid cancer (PTC) has been associated with resistance to 131 I. Our aim was to quantify the response to 131 I after surgery in patients who had the mutation (BRAF+) and those who did not have the mutated gene (BRAF-). A prospective cohort study was designed, from September 2015 to February 2016, which included patients with PTC receiving therapy after surgical treatment. Variables were described for age, gender, histology, tumor stage, thyroglobulin values before, 48h after and 6months after 131 I; absorbed dose and % activity on days 2 and 7 and elimination time. 41 patients giving in total 67 thyroid remnants were included. 61% were BRAF+. In stagesiii and iv, 80% were BRAF+. In lateral resection, 100% were BRAF+. The number of nodes was higher in BRAF+: 3.4 vs 1.2 (P=.01). The classic variant was predominant in BRAF+ (91.7% vs 8.3%, P=.03). 85.7% vs 14.3% of BRAF+ had desmoplastic reaction (P=.02). The BRAF+ had a lower absorbed dose than the administered activity (5.4Gy/MBq vs 20Gy/MBq, P=.02); lower% activity with respect to the unit of mass at 2 (0.046%/g vs 0.103%/g, P=.02) and at 7days (0.006%/gr vs 0.034%/gr, P=.04) CONCLUSIONS: The mutation of the BRAF V600E gene is related with greater resistance to postoperative treatment with 131 I since the onset of the disease. Copyright © 2018 AEC. Publicado por Elsevier España, S.L.U. All rights reserved.

Chronic lymphocytic thyroiditis and BRAF V600E in papillary thyroid carcinoma.

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Kim, Seo Ki; Woo, Jung-Woo; Lee, Jun Ho; Park, Inhye; Choe, Jun-Ho; Kim, Jung-Han; Kim, Jee Soo

2016-01-01

It has been reported that papillary thyroid carcinoma (PTC) with chronic lymphocytic thyroiditis (CLT) is less associated with extrathyroidal extension (ETE), advanced tumor stage and lymph node (LN) metastasis. Other studies have suggested that concurrent CLT could antagonize PTC progression, even in BRAF-positive patients. Since the clinical significance of the BRAF mutation has been particularly associated with conventional PTC, the purpose of this study was to determine the clinical significance of CLT according to BRAF mutation status in conventional PTC patients. We retrospectively reviewed the medical records of 3332 conventional PTC patients who underwent total thyroidectomy with bilateral central neck dissection at the Thyroid Cancer Center of Samsung Medical Center between January 2008 and June 2015. In this study, the prevalence of BRAF mutation was significantly less frequent in conventional PTC patients with CLT (76.9% vs 86.6%). CLT was an independent predictor for low prevalence of ETE in both BRAF-negative (OR=0.662, P=0.023) and BRAF-positive (OR=0.817, P=0.027) conventional PTC patients. In addition, CLT was an independent predictor for low prevalence of CLNM in both BRAF-negative (OR=0.675, P=0.044) and BRAF-positive (OR=0.817, P=0.030) conventional PTC patients. In conclusion, BRAF mutation was significantly less frequent in conventional PTC patients with CLT. However, CLT was an independent predictor for less aggressiveness in conventional PTC patients regardless of BRAF mutation status. © 2016 Society for Endocrinology.

Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues.

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Chat-Uthai, Nunthawut; Vejvisithsakul, Pichpisith; Udommethaporn, Sutthirat; Meesiri, Puttarakun; Danthanawanit, Chetiya; Wongchai, Yannawan; Teerapakpinyo, Chinachote; Shuangshoti, Shanop; Poungvarin, Naravat

2018-01-01

The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.

B-Raf mutation: a key player in molecular biology of cancer.

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Rahman, M A; Salajegheh, A; Smith, R A; Lam, A K-Y

2013-12-01

B-Raf is one of the more commonly mutated proto-oncogenes implicated in the development of cancers. In this review, we consider the mechanisms and clinical impacts of B-Raf mutations in cancer and discuss the implications for the patient in melanoma, thyroid cancer and colorectal cancer, where B-Raf mutations are particularly common. © 2013.

Multicenter Evaluation of a Novel Automated Rapid Detection System of BRAF Status in Formalin-Fixed, Paraffin-Embedded Tissues.

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Schiefer, Ana-Iris; Parlow, Laura; Gabler, Lisa; Mesteri, Ildiko; Koperek, Oskar; von Deimling, Andreas; Streubel, Berthold; Preusser, Matthias; Lehmann, Annika; Kellner, Udo; Pauwels, Patrick; Lambin, Suzan; Dietel, Manfred; Hummel, Michael; Klauschen, Frederick; Birner, Peter; Möbs, Markus

2016-05-01

The mutated BRAF oncogene represents a therapeutic target in malignant melanoma. Because BRAF mutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAF mutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAF V600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAF V600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Prediction of BRAF mutation status of craniopharyngioma using magnetic resonance imaging features.

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Yue, Qi; Yu, Yang; Shi, Zhifeng; Wang, Yongfei; Zhu, Wei; Du, Zunguo; Yao, Zhenwei; Chen, Liang; Mao, Ying

2017-10-06

OBJECTIVE Treatment with a BRAF mutation inhibitor might shrink otherwise refractory craniopharyngiomas and is a promising preoperative treatment to facilitate tumor resection. The aim of this study was to investigate the noninvasive diagnosis of BRAF-mutated craniopharyngiomas based on MRI characteristics. METHODS Fifty-two patients with pathologically diagnosed craniopharyngioma were included in this study. Polymerase chain reaction was performed on tumor tissue specimens to detect BRAF and CTNNB1 mutations. MRI manifestations-including tumor location, size, shape, and composition; signal intensity of cysts; enhancement pattern; pituitary stalk morphology; and encasement of the internal carotid artery-were analyzed by 2 neuroradiologists blinded to patient identity and clinical characteristics, including BRAF mutation status. Results were compared between the BRAF-mutated and wild-type (WT) groups. Characteristics that were significantly more prevalent (p < 0.05) in the BRAF-mutated craniopharyngiomas were defined as diagnostic features. The minimum number of diagnostic features needed to make a diagnosis was determined by analyzing the receiver operating characteristic (ROC) curve. RESULTS Eight of the 52 patients had BRAF-mutated craniopharyngiomas, and the remaining 44 had BRAF WT tumors. The clinical characteristics did not differ significantly between the 2 groups. Interobserver agreement for MRI data analysis was relatively reliable, with values of Cohen κ ranging from 0.65 to 0.97 (p < 0.001). A comparison of findings in the 2 patient groups showed that BRAF-mutated craniopharyngiomas tended to be suprasellar (p < 0.001), spherical (p = 0.005), predominantly solid (p = 0.003), and homogeneously enhancing (p < 0.001), and that patients with these tumors tended to have a thickened pituitary stalk (p = 0.014). When at least 3 of these 5 features were present, a tumor might be identified as BRAF mutated with a sensitivity of 1.00 and a specificity of 0

PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers.

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Filip Janku

Full Text Available Oncogenic mutations of PIK3CA, RAS (KRAS, NRAS, and BRAF have been identified in various malignancies, and activate the PI3K/AKT/mTOR and RAS/RAF/MEK pathways, respectively. Both pathways are critical drivers of tumorigenesis.Tumor tissues from 504 patients with diverse cancers referred to the Clinical Center for Targeted Therapy at MD Anderson Cancer Center starting in October 2008 were analyzed for PIK3CA, RAS (KRAS, NRAS, and BRAF mutations using polymerase chain reaction-based DNA sequencing.PIK3CA mutations were found in 54 (11% of 504 patients tested; KRAS in 69 (19% of 367; NRAS in 19 (8% of 225; and BRAF in 31 (9% of 361 patients. PIK3CA mutations were most frequent in squamous cervical (5/14, 36%, uterine (7/28, 25%, breast (6/29, 21%, and colorectal cancers (18/105, 17%; KRAS in pancreatic (5/9, 56%, colorectal (49/97, 51%, and uterine cancers (3/20, 15%; NRAS in melanoma (12/40, 30%, and uterine cancer (2/11, 18%; BRAF in melanoma (23/52, 44%, and colorectal cancer (5/88, 6%. Regardless of histology, KRAS mutations were found in 38% of patients with PIK3CA mutations compared to 16% of patients with wild-type (wtPIK3CA (p = 0.001. In total, RAS (KRAS, NRAS or BRAF mutations were found in 47% of patients with PIK3CA mutations vs. 24% of patients wtPIK3CA (p = 0.001. PIK3CA mutations were found in 28% of patients with KRAS mutations compared to 10% with wtKRAS (p = 0.001 and in 20% of patients with RAS (KRAS, NRAS or BRAF mutations compared to 8% with wtRAS (KRAS, NRAS or wtBRAF (p = 0.001.PIK3CA, RAS (KRAS, NRAS, and BRAF mutations are frequent in diverse tumors. In a wide variety of tumors, PIK3CA mutations coexist with RAS (KRAS, NRAS and BRAF mutations.

KIAA1549-BRAF fusions and IDH mutations can coexist in diffuse gliomas of adults.

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Badiali, Manuela; Gleize, Vincent; Paris, Sophie; Moi, Loredana; Elhouadani, Selma; Arcella, Antonietta; Morace, Roberta; Antonelli, Manila; Buttarelli, Francesca Romana; Figarella-Branger, Dominique; Kim, Young-Ho; Ohgaki, Hiroko; Mokhtari, Karima; Sanson, Marc; Giangaspero, Felice

2012-11-01

KIAA1549-BRAF fusion gene and isocitrate dehydrogenase (IDH) mutations are considered two mutually exclusive genetic events in pilocytic astrocytomas and diffuse gliomas, respectively. We investigated the presence of the KIAA1549-BRAF fusion gene in conjunction with IDH mutations and 1p/19q loss in 185 adult diffuse gliomas. Moreover BRAF(v600E) mutation was also screened. The KIAA1549-BRAF fusion gene was evaluated by reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. We found IDH mutations in 125 out 175 cases (71.4%). There were KIAA1549-BRAF fusion gene in 17 out of 180 (9.4%) cases and BRAF(v600E) in 2 out of 133 (1.5%) cases. In 11 of these 17 cases, both IDH mutations and the KIAA1549-BRAF fusion were present, as independent molecular events. Moreover, 6 of 17 cases showed co-presence of 1p/19q loss, IDH mutations and KIAA1549-BRAF fusion. Among the 17 cases with KIAA1549-BRAF fusion gene 15 (88.2%) were oligodendroglial neoplasms. Similarly, the two cases with BRAF(v600E) mutation were both oligodendroglioma and one had IDH mutations and 1p/19q co-deletion. Our results suggest that in a small fraction of diffuse gliomas, KIAA1549-BRAF fusion gene and BRAF(v600E) mutation may be responsible for deregulation of the Ras-RAF-ERK signaling pathway. Such alterations are more frequent in oligodendroglial neoplasm and may be co-present with IDH mutations and 1p/19q loss. © 2012 The Authors; Brain Pathology © 2012 International Society of Neuropathology.

An oncogenic mutant of RHEB, RHEB Y35N, exhibits an altered interaction with BRAF resulting in cancer transformation.

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Heard, Jeffrey J; Phung, Ivy; Potes, Mark I; Tamanoi, Fuyuhiko

2018-01-10

RHEB is a unique member of the RAS superfamily of small GTPases expressed in all tissues and conserved from yeast to humans. Early studies on RHEB indicated a possible RHEB-RAF interaction, but this has not been fully explored. Recent work on cancer genome databases has revealed a reoccurring mutation in RHEB at the Tyr35 position, and a recent study points to the oncogenic potential of this mutant that involves activation of RAF/MEK/ERK signaling. These developments prompted us to reassess the significance of RHEB effect on RAF, and to compare mutant and wild type RHEB. To study RHEB-RAF interaction, and the effect of the Y35N mutation on this interaction, we used transfection, immunoprecipitation, and Western blotting techniques. We generated cell lines stably expressing RHEB WT, RHEB Y35N, and KRAS G12V, and monitored cellular transforming properties through cell proliferation, anchorage independent growth, cell cycle analysis, and foci formation assays. We observe a strong interaction between RHEB and BRAF, but not with CRAF. This interaction is dependent on an intact RHEB effector domain and RHEB-GTP loading status. RHEB overexpression decreases RAF activation of the RAF/MEK/ERK pathway and RHEB knockdown results in an increase in RAF/MEK/ERK activation. RHEB Y35N mutation has decreased interaction with BRAF, and RHEB Y35N cells exhibit greater BRAF/CRAF heterodimerization resulting in increased RAF/MEK/ERK signaling. This leads to cancer transformation of RHEB Y35N stably expressing cell lines, similar to KRAS G12 V expressing cell lines. RHEB interaction with BRAF is crucial for inhibiting RAF/MEK/ERK signaling. The RHEB Y35N mutant sustains RAF/MEK/ERK signaling due to a decreased interaction with BRAF, leading to increased BRAF/CRAF heterodimerization. RHEB Y35N expressing cells undergo cancer transformation due to decreased interaction between RHEB and BRAF resulting in overactive RAF/MEK/ERK signaling. Taken together with the previously established

Growth-Inhibitory and Antiangiogenic Activity of the MEK Inhibitor PD0325901 in Malignant Melanoma with or without BRAF Mutations

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Ludovica Ciuffreda

2009-08-01

Full Text Available The Raf/MEK/ERK pathway is an importantmediator of tumor cell proliferation and angiogenesis. Here, weinvestigated the growth-inhibitory and antiangiogenic properties of PD0325901, a novel MEK inhibitor, in human melanoma cells. PD0325901 effects were determined in a panel of melanoma cell lines with different genetic aberrations. PD0325901 markedly inhibited ERK phosphorylation and growth of both BRAF mutant and wild-type melanoma cell lines, with IC50 in the nanomolar range even in the least responsive models. Growth inhibition was observed both in vitro and in vivo in xenograft models, regardless of BRAF mutation status, and was due to G1-phase cell cycle arrest and subsequent induction of apoptosis. Cell cycle (cyclin D1, c-Myc, and p27KIP1 and apoptosis (Bcl-2 and survivin regulators were modulated by PD0325901 at the protein level. Gene expression profiling revealed profound modulation of several genes involved in the negative control of MAPK signaling and melanoma cell differentiation, suggesting alternative, potentially relevant mechanisms of action. Finally, PD0325901 inhibited the production of the proangiogenic factors vascular endothelial growth factor and interleukin 8 at a transcriptional level. In conclusion, PD0325901 exerts potent growth-inhibitory, proapoptotic, and antiangiogenic activity in melanoma lines, regardless of their BRAF mutation status. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective treatment strategies for patients experiencing malignant melanoma.

BRAF mutation is not predictive of long-term outcome in papillary thyroid carcinoma

International Nuclear Information System (INIS)

Henke, Lauren E; Pfeifer, John D; Ma, Changquing; Perkins, Stephanie M; DeWees, Todd; El-Mofty, Samir; Moley, Jeffrey F; Nussenbaum, Brian; Haughey, Bruce H; Baranski, Thomas J; Schwarz, Julie K; Grigsby, Perry W

2015-01-01

The BRAF mutation occurs commonly in papillary thyroid carcinoma (PTC). Previous investigations of its utility to predict recurrence-free survival (RFS) and disease-specific survival (DSS) have reported conflicting results and its role remains unclear. The purpose of this retrospective study was to determine the incidence of the BRAF mutation and analyze its relationship to clinicopathologic risk factors and long-term outcomes in the largest, single-institution American cohort to date. BRAF mutational status was determined in 508 PTC patients using RFLP analysis. The relationships between BRAF mutation status, patient and tumor characteristics, RFS, and DSS were analyzed. The BRAF mutation was present in 67% of patients. On multivariate analysis, presence of the mutation predicted only for capsular invasion (HR, 1.7; 95% CI, 1.1–2.6), cervical lymph node involvement (HR, 1.7; 95% CI, 1.1–2.7), and classic papillary histology (HR, 1.8; 95% CI 1.1–2.9). There was no significant relationship between the BRAF mutation and RFS or DSS, an observation that was consistent across univariate, multivariate, and Kaplan–Meier analyses. This is the most extensive study to date in the United States to demonstrate that BRAF mutation is of no predictive value for recurrence or survival in PTC. We found correlations of BRAF status and several clinicopathologic characteristics of high-risk disease, but limited evidence that the mutation correlates with more extensive or aggressive disease. This analysis suggests that BRAF is minimally prognostic in PTC. However, prevalence of the BRAF mutation is 70% in the general population, providing the opportunity for targeted therapy

Behandlingen af BRAF-muteret metastatisk melanom med BRAF/MEK-inhibitorer bedre prognosen

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Boyles, Tessa Bystrup; Svane, Inge Marie; Bastholt, Lars

2016-01-01

Melanoma is an aggressive form of skin cancer which is the cause of a great number of skin cancer-related deaths worldwide and about 300 deaths in Denmark. After several years of failure of treatment of metastatic melanoma, the development of BRAF- and later MEK inhibitors was considered...

Behandlingen af BRAF-muteret metastatisk melanom med BRAF/MEK-inhibitorer bedrer prognosen

DEFF Research Database (Denmark)

Boyles, Tessa Bystrup; Svane, Inge Marie; Bastholt, Lars

2016-01-01

Melanoma is an aggressive form of skin cancer which is the cause of a great number of skin cancer-related deaths worldwide and about 300 deaths in Denmark. After several years of failure of treatment of metastatic melanoma, the development of BRAF- and later MEK inhibitors was considered...

Dysfunctional oxidative phosphorylation makes malignant melanoma cells addicted to glycolysis driven by the (V600E)BRAF oncogene

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Hall, Arnaldur; Meyle, Kathrine Damm; Lange, Marina Krarup

2013-01-01

basis for this addiction is largely unknown. Here we provide evidence for a metabolic rationale behind the addiction to (V600E)BRAF in two malignant melanoma cell lines. Both cell lines display a striking addiction to glycolysis due to underlying dysfunction of oxidative phosphorylation (OXPHOS......). Notably, even minor reductions in glycolytic activity lead to increased OXPHOS activity (reversed Warburg effect), however the mitochondria are unable to sustain ATP production. We show that (V600E)BRAF upholds the activity of glycolysis and therefore the addiction to glycolysis de facto becomes...

BRAF mutations in conjunctival melanoma: investigation of incidence, clinicopathological features, prognosis and paired premalignant lesions

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Larsen, Ann-Cathrine; Dahl, Christina; Dahmcke, Christina M.

2016-01-01

with atypia. BRAF mutations were identified in 39 of 111 (35%) cases. The rate ratio of BRAF-mutated versus BRAF-wild-type melanoma did not change over time. BRAF mutations were associated with T1 stage (p = 0.007), young age (p = 0.001), male gender (p = 0.02), sun-exposed location (p = 0.01), mixed....../non-pigmented tumour colour (p = 0.02) and nevus origin (p = 0.005), but did not associate with prognosis. BRAF status in conjunctival melanoma and paired premalignant lesions corresponded in 19 of 20 cases. Immunohistochemistry detected BRAF V600E mutations with a sensitivity of 0.94 and a specificity of 1...

Associations of MC1R Genotype and Patient Phenotypes with BRAF and NRAS Mutations in Melanoma.

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Thomas, Nancy E; Edmiston, Sharon N; Kanetsky, Peter A; Busam, Klaus J; Kricker, Anne; Armstrong, Bruce K; Cust, Anne E; Anton-Culver, Hoda; Gruber, Stephen B; Luo, Li; Orlow, Irene; Reiner, Anne S; Gallagher, Richard P; Zanetti, Roberto; Rosso, Stefano; Sacchetto, Lidia; Dwyer, Terence; Parrish, Eloise A; Hao, Honglin; Gibbs, David C; Frank, Jill S; Ollila, David W; Begg, Colin B; Berwick, Marianne; Conway, Kathleen

2017-12-01

Associations of MC1R with BRAF mutations in melanoma have been inconsistent between studies. We sought to determine for 1,227 participants in the international population-based Genes, Environment, and Melanoma (GEM) study whether MC1R and phenotypes were associated with melanoma BRAF/NRAS subtypes. We used logistic regression adjusted by age, sex, and study design features and examined effect modifications. BRAF + were associated with younger age, blond/light brown hair, increased nevi, and less freckling, and NRAS + with older age relative to the wild type (BRAF - /NRAS - ) melanomas (all P < 0.05). Comparing specific BRAF subtypes to the wild type, BRAF V600E was associated with younger age, blond/light brown hair, and increased nevi and V600K with increased nevi and less freckling (all P < 0.05). MC1R was positively associated with BRAF V600E cases but only among individuals with darker phototypes or darker hair (P interaction < 0.05) but inversely associated with BRAF V600K (P trend  = 0.006) with no significant effect modification by phenotypes. These results support distinct etiologies for BRAF V600E, BRAF V600K, NRAS + , and wild-type melanomas. MC1R's associations with BRAF V600E cases limited to individuals with darker phenotypes indicate that MC1R genotypes specifically provide information about BRAF V600E melanoma risk in those not considered high risk based on phenotype. Our results also suggest that melanin pathways deserve further study in BRAF V600E melanomagenesis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

BRAF V600E mutations in papillary craniopharyngioma

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Brastianos, Priscilla K.; Santagata, Sandro

2016-01-01

Papillary craniopharyngioma is an intracranial tumor that results in high levels of morbidity. We recently demonstrated that the vast majority of these tumors harbor the oncogenic BRAF V600E mutation. The pathologic diagnosis of papillary craniopharyngioma can now be confirmed using mutation specific immunohistochemistry and targeted genetic testing. Treatment with targeted agents is now also a possibility in select situations. We recently reported a patient with a multiply recurrent papillary craniopharyngioma in whom targeting both BRAF and MEK resulted in a dramatic therapeutic response with a marked anti-tumor immune response. This work shows that activation of the MAPK pathway is the likely principal oncogenic driver of these tumors. We will now investigate the efficacy of this approach in a multicenter phase II clinical trial. Post-treatment resection samples will be monitored for the emergence of resistance mechanisms. Further advances in the non-invasive diagnosis of papillary craniopharyngioma by radiologic criteria and by cell-free DNA testing could someday allow neo-adjuvant therapy for this disease in select patient populations. PMID:26563980

BRAF mutation in papillary thyroid microcarcinoma – additional marker of risk stratification

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Dmitriy Yuriyevich Semyonov

2014-11-01

Full Text Available BackgroundPapillary thyroid microcarcinoma (PTMC is heterogeneous group of tumor less than 1 cm in the diameter. The volume of surgical treatment stay unstable because unclear biological potential of PTMC.AimThe aim of our study was to assess the utility of BRAF gene mutation as preoperative additional marker of risk stratification.Materials and methodsWe include 44 patient who were operated in general surgery department Pavlov State Medical University from 2001 to 2013. In all 44 cases BRAF gene mutation was detected and compared with clinic-morphological features (multifocality, invasive growth, lymph node metastasis, recurrence retrospectivelyResultsIn our study the frequency of BRAF gene mutation was 68.2%. On multivariate regression analysis the presence of bilateral tumoural foci, lymph node metastasis and the presence of capsular invasion were significantly related to BRAF positive gene status.ConclusionsThus, appropriate volume for the BRAF positive PTMC is thyroidectomy with central compartment lymph node dissection.

Detecting BRAF Mutations in Formalin-Fixed Melanoma: Experiences with TwoState-of-the-Art Techniques

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Nicola L. Schoenewolf

2012-06-01

Full Text Available Background: Melanoma is characterized by a high frequency of BRAF mutations. It is unknown if the BRAF mutation status has any predictive value for therapeutic approaches such as angiogenesis inhibition. Patients and Methods: We used 2 methods to analyze the BRAF mutation status in 52 of 62 melanoma patients. Method 1 (mutation-specific real-time PCR specifically detects the most frequent BRAF mutations, V600E and V600K. Method 2 (denaturing gel gradient electrophoresis and direct sequencing identifies any mutations affecting exons 11 and 15. Results: Eighteen BRAF mutations and 15 wild-type mutations were identified with both methods. One tumor had a double mutation (GAA in codon 600. Results of 3 samples were discrepant. Additional mutations (V600M, K601E were detected using method 2. Sixteen DNA samples were analyzable with either method 1 or method 2. There was a significant association between BRAF V600E mutation and survival. Conclusion: Standardized tissue fixation protocols are needed to optimize BRAF mutation analysis in melanoma. For melanoma treatment decisions, the availability of a fast and reliable BRAF V600E screening method may be sufficient. If other BRAF mutations in exons 11 and 15 are found to be of predictive value, a combination of the 2 methods would be useful.

Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells

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Luca Mologni

2018-05-01

Full Text Available BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAFV600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion within an amplified BRAFV600E locus and a missense point mutation of the transcriptional repressor BCORL1 in vemurafenib-resistant A375 melanoma cells. Functional data confirmed that truncated p47BRAFV600E and mutant BCORL1Q1076H both contribute to resistance. Interestingly, either endogenous BCORL1 silencing or ectopic BCORL1Q1076H expression mimicked the effects of a CRISPR/Cas9-edited BCORL1Q1076H locus, suggesting a complex mixture of loss- and gain-of-function effects caused by the mutation. Transcriptomic data confirmed this hypothesis. Finally, we show that the pan-RAF inhibitor sorafenib is not affected by expression of BRAF deletion variant and effectively synergizes with vemurafenib to block resistant cells, suggesting a possible intervention for this class of mutants.

The distribution of BRAF gene fusions in solid tumors and response to targeted therapy.

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Ross, Jeffrey S; Wang, Kai; Chmielecki, Juliann; Gay, Laurie; Johnson, Adrienne; Chudnovsky, Jacob; Yelensky, Roman; Lipson, Doron; Ali, Siraj M; Elvin, Julia A; Vergilio, Jo-Anne; Roels, Steven; Miller, Vincent A; Nakamura, Brooke N; Gray, Adam; Wong, Michael K; Stephens, Philip J

2016-02-15

Although the BRAF V600E base substitution is an approved target for the BRAF inhibitors in melanoma, BRAF gene fusions have not been investigated as anticancer drug targets. In our study, a wide variety of tumors underwent comprehensive genomic profiling for hundreds of known cancer genes using the FoundationOne™ or FoundationOne Heme™ comprehensive genomic profiling assays. BRAF fusions involving the intact in-frame BRAF kinase domain were observed in 55 (0.3%) of 20,573 tumors, across 12 distinct tumor types, including 20 novel BRAF fusions. These comprised 29 unique 5' fusion partners, of which 31% (9) were known and 69% (20) were novel. BRAF fusions included 3% (14/531) of melanomas; 2% (15/701) of gliomas; 1.0% (3/294) of thyroid cancers; 0.3% (3/1,062) pancreatic carcinomas; 0.2% (8/4,013) nonsmall-cell lung cancers and 0.2% (4/2,154) of colorectal cancers, and were enriched in pilocytic (30%) vs. nonpilocytic gliomas (1%; p < 0.0001), Spitzoid (75%) vs. nonSpitzoid melanomas (1%; p = 0.0001), acinar (67%) vs. nonacinar pancreatic cancers (<1%; p < 0.0001) and papillary (3%) vs. nonpapillary thyroid cancers (0%; p < 0.03). Clinical responses to trametinib and sorafenib are presented. In conclusion, BRAF fusions are rare driver alterations in a wide variety of malignant neoplasms, but enriched in Spitzoid melanoma, pilocytic astrocytomas, pancreatic acinar and papillary thyroid cancers. © 2015 The Authors. Published by Wiley Periodicals, Inc. on behalf of UICC.

High BRAF Mutation Frequency and Marked Survival Differences in Subgroups According to KRAS/BRAF Mutation Status and Tumor Tissue Availability in a Prospective Population-Based Metastatic Colorectal Cancer Cohort

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Sorbye, Halfdan; Dragomir, Anca; Sundström, Magnus

2015-01-01

were analyzed in a prospectively collected unselected population-based cohort of 798 non-resectable mCRC patients. The cohort contained many patients with poor performance status (39% PS 2-4) and elderly (37% age>75), groups usually not included in clinical trials. Patients without available tissue...... patients. Median survival in this cohort varied from 1 month in BRAF mutated patients not given chemotherapy to 26 months in wildtype KRAS/BRAF patients availability, BRAF mutation and KRAS mutation were all independent prognostic factors for survival. The observed 21% BRAF......CRC patients. Survival in unselected metastatic colorectal cancer patients is extremely variable and subgroups have an extremely short survival compared to trial patients. Patients without available TMA had worse prognostic factors and shorter survival, which questions the total generalizability of present TMA...

BRAF V600E mutational status in bile duct adenomas and hamartomas.

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Pujals, Anaïs; Bioulac-Sage, Paulette; Castain, Claire; Charpy, Cécile; Zafrani, Elie Serge; Calderaro, Julien

2015-10-01

Bile duct adenomas (BDA) and bile duct hamartomas (BDH) are benign bile duct lesions considered neoplastic or secondary to ductal plate malformation, respectively. We have reported previously a high prevalence of BRAF V600E mutations detected by allele-specific polymerase chain reaction assay in BDA, and suggested that BDA may be precursors to a subset of intrahepatic cholangiocarcinomas harbouring V600E mutations. The aim of the present study was to assess the existence of BRAF V600E mutations, using immunohistochemical methods, in additional BDA as well as in BDH. Fifteen BDA and 35 BDH were retrieved from the archives of the pathology departments of two French university hospitals. All cases were reviewed by two pathologists specialized in liver diseases. BRAF V600E mutational status was investigated by immunohistochemistry. Mutated BRAF mutant protein was detected in 53% of the BDA and in none of the cases of BDH. Our findings suggest that BDA and BDH are different processes, and that BDA represent true benign neoplasms. They also support the hypothesis that mutated BDA might precede the development of the subset of intrahepatic cholangiocarcinomas harbouring BRAF V600E mutations. © 2015 John Wiley & Sons Ltd.

Frequency of BRAF V600E Mutation in the Mexican Population of Patients With Metastatic Melanoma

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Erika Ruiz-Garcia

2017-06-01

Full Text Available Purpose: The BRAF V600E mutation has been described in melanomas occurring in the Caucasian, European, and Asian populations. However, in the Mexican population, the status and clinical significance of BRAF mutation has not been researched on a large scale. Methods: Consecutive BRAF-tested Mexican patients with metastatic melanoma (n = 127 were analyzed for mutations in exon 15 of the BRAF gene in genomic DNA by real-time polymerase chain reaction technology for amplification and detection. The results were correlated with the clinical-pathologic features and the prognosis of the patients. Results: The frequency of somatic mutation V600E within the BRAF gene was 54.6% (43 of 127 patients. Nodular melanoma was the most prevalent subtype in our population, with BRAF mutations in 37.2% (16 of 55 patients. In contrast, superficial spread had a frequency of 18.6% BRAF mutation (eight of 24. Other clinicopathologic features were assessed to correlate with the mutation status. Conclusion: This study searched for the most prevalent BRAF V600E mutation type in melanoma in a heterogeneous population from Mexico. Nodular melanoma was found to be the most prevalent in metastatic presentation and the presence of BRAF V600E mutation, perhaps related to the mixed ancestry; in the north, ancestry is predominantly European and in the south, it is predominantly Asian. The outcomes of the mutation correlations were similar to those found in other populations.

BRAF inhibitors and radiotherapy for melanoma brain metastases: potential advantages and disadvantages of combination therapy

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Chowdhary M

2016-12-01

Full Text Available Mudit Chowdhary,1,2 Kirtesh R Patel,1 Hasan H Danish,1 David H Lawson,3 Mohammad K Khan1 1Department of Radiation Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, 2Department of Radiation Oncology, Rush University Medical Center, Chicago, IL, 3Department of Hematology and Medical Oncology, Winship Cancer Institute, Emory University, Atlanta, GA, USA Abstract: Melanoma is an aggressive malignancy that frequently spreads to the brain, resulting in rapid deterioration in both quality and quantity of life. Historically, treatment options for melanoma brain metastases (MBM have predominantly consisted of surgery and radiotherapy. While these options can help provide local control, the majority of patients still develop intracranial progression. Indeed, novel therapeutic options, including molecularly targeted agents and immunotherapy, have improved outcomes and are now changing the role of radiotherapy. Up to 50% of melanomas contain an activating BRAF mutation, resulting in hyperactive cellular proliferation and survival. Drugs that target BRAF have been introduced for the treatment of metastatic melanoma and offer hope in improving disease outcomes; however, many of these trials either excluded or had a limited amount of patients with MBM. Recent studies have revealed that melanoma cell lines become more radiosensitive following BRAF inhibition, thus providing a potential synergistic mechanism when combining BRAF inhibitor (BRAFi and radiotherapy. However, neurotoxicity concerns also exist with this combination. This article reviews the efficacy and limitations of BRAFi therapy for MBM, describes current evidence for combining BRAFis with radiation, discusses the rationale and evidence for combination modalities, and highlights emerging clinical trials specifically investigating this combination in MBM. Keywords: brain metastases, melanoma, radiation, BRAF inhibitors, vemurafenib, dabrafenib

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

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Nicolas Piton

2015-01-01

Full Text Available KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27% and specificity (64% in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100% and specificity (100% in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer.

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

Science.gov (United States)

Borrini, Francesco; Bolognese, Antonio; Lamy, Aude; Sabourin, Jean-Christophe

2015-01-01

KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR) monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE) samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27%) and specificity (64%) in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100%) and specificity (100%) in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer. PMID:25983749

Ablation of BRaf impairs neuronal differentiation in the postnatal hippocampus and cerebellum.

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Verena Pfeiffer

Full Text Available This study focuses on the role of the kinase BRaf in postnatal brain development. Mice expressing truncated, non-functional BRaf in neural stem cell-derived brain tissue demonstrate alterations in the cerebellum, with decreased sizes and fuzzy borders of the glomeruli in the granule cell layer. In addition we observed reduced numbers and misplaced ectopic Purkinje cells that showed an altered structure of their dendritic arborizations in the hippocampus, while the overall cornus ammonis architecture appeared to be unchanged. In male mice lacking BRaf in the hippocampus the size of the granule cell layer was normal at postnatal day 12 (P12 but diminished at P21, as compared to control littermates. This defect was caused by a reduced ability of dentate gyrus progenitor cells to differentiate into NeuN positive granule cell neurons. In vitro cell culture of P0/P1 hippocampal cells revealed that BRaf deficient cells were impaired in their ability to form microtubule-associated protein 2 positive neurons. Together with the alterations in behaviour, such as autoaggression and loss of balance fitness, these observations indicate that in the absence of BRaf all neuronal cellular structures develop, but neuronal circuits in the cerebellum and hippocampus are partially disturbed besides impaired neuronal generation in both structures.

Dabrafenib plus trametinib in patients with previously untreated BRAF(V600E)-mutant metastatic non-small-cell lung cancer : An open-label, phase 2 trial

NARCIS (Netherlands)

Planchard, David; Smit, Egbert F.; Groen, Harry J. M.; Mazieres, Julien; Besse, Benjamin; Helland, Aslaug; Giannone, Vanessa; D'Amelio, Anthony M.; Zhang, Pingkuan; Mookerjee, Bijoyesh; Johnson, Bruce E.

2017-01-01

Background: BRAF(V600E) mutation occurs in 1-2% of lung adenocarcinomas and acts as an oncogenic driver. Dabrafenib, alone or combined with trametinib, has shown substantial antitumour activity in patients with previously treated BRAF(V600E)-mutant metastatic non-small-cell lung cancer (NSCLC). We

Association between dermoscopic and reflectance confocal microscopy features of cutaneous melanoma with BRAF mutational status.

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Bombonato, C; Ribero, S; Pozzobon, F C; Puig-Butille, J A; Badenas, C; Carrera, C; Malvehy, J; Moscarella, E; Lallas, A; Piana, S; Puig, S; Argenziano, G; Longo, C

2017-04-01

Melanomas harbouring common genetic mutations might share certain morphological features detectable with dermoscopy and reflectance confocal microscopy. BRAF mutational status is crucial for the management of metastatic melanoma. To correlate the dermoscopic characteristics of primary cutaneous melanomas with BRAF mutational status. Furthermore, a subset of tumours has also been analysed for the presence of possible confocal features that might be linked with BRAF status. Retrospectively acquired dermoscopic and confocal images of patients with melanoma in tertiary referral academic centres: Skin Cancer Unit in Reggio Emilia and at the Melanoma Unit in Barcelona. Kruskal-Wallis test, logistic regressions, univariate and multivariate analyses have been performed to find dermoscopic and confocal features significantly correlated with BRAF mutational status. Dermoscopically, the presence of irregular peripheral streaks and ulceration were positive predictors of BRAF-mutated melanomas with a statistically significance value, while dotted vessels were more represented in wild-type melanomas. None of the evaluated reflectance confocal microscopy features were correlated with genetic profiling. Ulceration and irregular peripheral streaks represent dermoscopic feature indicative for BRAF-mutated melanoma, while dotted vessels are suggestive for wild-type melanoma. © 2016 European Academy of Dermatology and Venereology.

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH.

Science.gov (United States)

Yun, Jihye; Mullarky, Edouard; Lu, Changyuan; Bosch, Kaitlyn N; Kavalier, Adam; Rivera, Keith; Roper, Jatin; Chio, Iok In Christine; Giannopoulou, Eugenia G; Rago, Carlo; Muley, Ashlesha; Asara, John M; Paik, Jihye; Elemento, Olivier; Chen, Zhengming; Pappin, Darryl J; Dow, Lukas E; Papadopoulos, Nickolas; Gross, Steven S; Cantley, Lewis C

2015-12-11

More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations and are often refractory to approved targeted therapies. We found that cultured human CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C, depleting glutathione. Thus, reactive oxygen species accumulate and inactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibition of GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. High-dose vitamin C impairs tumor growth in Apc/Kras(G12D) mutant mice. These results provide a mechanistic rationale for exploring the therapeutic use of vitamin C for CRCs with KRAS or BRAF mutations. Copyright © 2015, American Association for the Advancement of Science.

Multifocal fibrosing thyroiditis and its association with papillary thyroid carcinoma using BRAF pyrosequencing.

Science.gov (United States)

Frank, Renee; Baloch, Zubair W; Gentile, Caren; Watt, Christopher D; LiVolsi, Virginia A

2014-09-01

Multifocal fibrosing thyroiditis (MFT) is characterized by numerous foci of fibrosis in a stellate configuration with fibroelastotic and fibroblastic centers entrapping epithelial structures. MFT has been proposed as a risk factor for papillary thyroid carcinoma (PTC) development. We attempted to identify whether MFT showed such molecular changes and could possibly be related to PTC. We identified seven cases of PTC with MFT in our institutional pathology database and personal consult service of one of the authors (VAL) for the years 1999 to 2012. Areas of PTC, MFT, and normal tissue were selected for BRAF analysis. Macro-dissection, DNA extraction and PCR amplification, and pyrosequencing were performed to detect BRAF mutations in codon 600. All of the MFT lesions and normal thyroid tissue were negative for BRAF mutations. Of the seven PTCs analyzed, five (71 %) were negative for BRAF mutations, while two cases were positive. In our study, none of the MFT lesions harbored BRAF mutations, whereas 29 % (two of seven) PTCs in the same gland were positive. Hence, in this small study, we found no evidence that the MFT lesion is a direct precursor to PTC. It is likely an incidental bystander in the process and a reflection of the background thyroiditis.

Dramatic Response of BRAF V600E Mutant Papillary Craniopharyngioma to Targeted Therapy.

Science.gov (United States)

Brastianos, Priscilla K; Shankar, Ganesh M; Gill, Corey M; Taylor-Weiner, Amaro; Nayyar, Naema; Panka, David J; Sullivan, Ryan J; Frederick, Dennie T; Abedalthagafi, Malak; Jones, Pamela S; Dunn, Ian F; Nahed, Brian V; Romero, Javier M; Louis, David N; Getz, Gad; Cahill, Daniel P; Santagata, Sandro; Curry, William T; Barker, Fred G

2016-02-01

We recently reported that BRAF V600E is the principal oncogenic driver of papillary craniopharyngioma, a highly morbid intracranial tumor commonly refractory to treatment. Here, we describe our treatment of a man age 39 years with multiply recurrent BRAF V600E craniopharyngioma using dabrafenib (150mg, orally twice daily) and trametinib (2mg, orally twice daily). After 35 days of treatment, tumor volume was reduced by 85%. Mutations that commonly mediate resistance to MAPK pathway inhibition were not detected in a post-treatment sample by whole exome sequencing. A blood-based BRAF V600E assay detected circulating BRAF V600E in the patient's blood. Re-evaluation of the existing management paradigms for craniopharyngioma is warranted, as patient morbidity might be reduced by noninvasive mutation testing and neoadjuvant-targeted treatment. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

First-Line Cetuximab Monotherapy in KRAS/NRAS/BRAF Mutation-Negative Colorectal Cancer Patients.

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Moiseyenko, Vladimir M; Moiseyenko, Fedor V; Yanus, Grigoriy A; Kuligina, Ekatherina Sh; Sokolenko, Anna P; Bizin, Ilya V; Kudriavtsev, Alexey A; Aleksakhina, Svetlana N; Volkov, Nikita M; Chubenko, Vyacheslav A; Kozyreva, Kseniya S; Kramchaninov, Mikhail M; Zhuravlev, Alexandr S; Shelekhova, Kseniya V; Pashkov, Denis V; Ivantsov, Alexandr O; Venina, Aigul R; Sokolova, Tatyana N; Preobrazhenskaya, Elena V; Mitiushkina, Natalia V; Togo, Alexandr V; Iyevleva, Aglaya G; Imyanitov, Evgeny N

2018-06-01

Colorectal carcinomas (CRCs) are sensitive to treatment by anti-epidermal growth factor receptor (EGFR) antibodies only if they do not carry activating mutations in down-stream EGFR targets (KRAS/NRAS/BRAF). Most clinical trials for chemo-naive CRC patients involved combination of targeted agents and chemotherapy, while single-agent cetuximab or panitumumab studies included either heavily pretreated patients or subjects who were not selected on the basis of molecular tests. We hypothesized that anti-EGFR therapy would have significant efficacy in chemo-naive patients with KRAS/NRAS/BRAF mutation-negative CRC. Nineteen patients were prospectively included in the study. Two (11%) patients experienced partial response (PR) and 11 (58%) subjects showed stable disease (SD). Median time to progression approached 6.1 months (range 1.6-15.0 months). Cetuximab efficacy did not correlate with RNA expression of EGFR and insulin-like growth factor 2 (IGF2). Only one tumor carried PIK3CA mutation, and this CRC responded to cetuximab. Exome analysis of patients with progressive disease (PD) revealed 1 CRC with high-level microsatellite instability and 1 instance of HER2 oncogene amplification; 3 of 4 remaining patients with PD had allergic reactions to cetuximab, while none of the subjects with PR or SD had this complication. Comparison with 19 retrospective KRAS/NRAS/BRAF mutation-negative patients receiving first-line fluoropyrimidines revealed no advantages or disadvantages of cetuximab therapy. Cetuximab demonstrates only modest efficacy when given as a first-line monotherapy to KRAS/NRAS/BRAF mutation-negative CRC patients. It is of question, why meticulous patient selection, which was undertaken in the current study, did not result in the improvement of outcomes of single-agent cetuximab treatment.

Detection of BRAF V600 mutations in melanoma: evaluation of concordance between the Cobas® 4800 BRAF V600 mutation test and the methods used in French National Cancer Institute (INCa) platforms in a real-life setting.

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Mourah, Samia; Denis, Marc G; Narducci, Fabienne Escande; Solassol, Jérôme; Merlin, Jean-Louis; Sabourin, Jean-Christophe; Scoazec, Jean-Yves; Ouafik, L'Houcine; Emile, Jean-François; Heller, Remy; Souvignet, Claude; Bergougnoux, Loïc; Merlio, Jean-Philippe

2015-01-01

Vemurafenib is approved for the treatment of metastatic melanoma in patients with BRAF V600 mutation. In pivotal clinical trials, BRAF testing has always been done with the approved cobas 4800 BRAF test. In routine practice, several methods are available and are used according to the laboratories usual procedures. A national, multicenter, non-interventional study was conducted with prospective and consecutive collection of tumor samples. A parallel evaluation was performed in routine practice between the cobas 4800 BRAF V600 mutation test and home brew methods (HBMs) of 12 national laboratories, labelled and funded by the French National Cancer Institute (INCa). For 420 melanoma samples tested, the cobas method versus HBM showed a high concordance (93.3%; kappa = 0.86) in BRAF V600 genotyping with similar mutation rates (34.0% versus 35.7%, respectively). Overall, 97.4% and 98.6% of samples gave valid results using the cobas and HBM, respectively. Of the 185 samples strictly fulfilling the cobas guidelines, the concordance rate was even higher (95.7%; kappa = 0.91; 95%CI [0.85; 0.97]). Out of the 420 samples tested, 28 (6.7%) showed discordance between HBM and cobas. This prospective study shows a high concordance rate between the cobas 4800 BRAF V600 test and home brew methods in the routine detection of BRAF V600E mutations.

Detection of BRAF V600 Mutations in Melanoma: Evaluation of Concordance between the Cobas® 4800 BRAF V600 Mutation Test and the Methods Used in French National Cancer Institute (INCa) Platforms in a Real-Life Setting

Science.gov (United States)

Mourah, Samia; Denis, Marc G.; Narducci, Fabienne Escande; Solassol, Jérôme; Merlin, Jean-Louis; Sabourin, Jean-Christophe; Scoazec, Jean-Yves; Ouafik, L’Houcine; Emile, Jean-François; Heller, Remy; Souvignet, Claude; Bergougnoux, Loïc; Merlio, Jean-Philippe

2015-01-01

Vemurafenib is approved for the treatment of metastatic melanoma in patients with BRAF V600 mutation. In pivotal clinical trials, BRAF testing has always been done with the approved cobas 4800 BRAF test. In routine practice, several methods are available and are used according to the laboratories usual procedures. A national, multicenter, non-interventional study was conducted with prospective and consecutive collection of tumor samples. A parallel evaluation was performed in routine practice between the cobas 4800 BRAF V600 mutation test and home brew methods (HBMs) of 12 national laboratories, labelled and funded by the French National Cancer Institute (INCa). For 420 melanoma samples tested, the cobas method versus HBM showed a high concordance (93.3%; kappa = 0.86) in BRAF V600 genotyping with similar mutation rates (34.0% versus 35.7%, respectively). Overall, 97.4% and 98.6% of samples gave valid results using the cobas and HBM, respectively. Of the 185 samples strictly fulfilling the cobas guidelines, the concordance rate was even higher (95.7%; kappa = 0.91; 95%CI [0.85; 0.97]). Out of the 420 samples tested, 28 (6.7%) showed discordance between HBM and cobas. This prospective study shows a high concordance rate between the cobas 4800 BRAF V600 test and home brew methods in the routine detection of BRAF V600E mutations. PMID:25789737

Detection of BRAF V600 mutations in melanoma: evaluation of concordance between the Cobas® 4800 BRAF V600 mutation test and the methods used in French National Cancer Institute (INCa platforms in a real-life setting.

Directory of Open Access Journals (Sweden)

Samia Mourah

Full Text Available Vemurafenib is approved for the treatment of metastatic melanoma in patients with BRAF V600 mutation. In pivotal clinical trials, BRAF testing has always been done with the approved cobas 4800 BRAF test. In routine practice, several methods are available and are used according to the laboratories usual procedures. A national, multicenter, non-interventional study was conducted with prospective and consecutive collection of tumor samples. A parallel evaluation was performed in routine practice between the cobas 4800 BRAF V600 mutation test and home brew methods (HBMs of 12 national laboratories, labelled and funded by the French National Cancer Institute (INCa. For 420 melanoma samples tested, the cobas method versus HBM showed a high concordance (93.3%; kappa = 0.86 in BRAF V600 genotyping with similar mutation rates (34.0% versus 35.7%, respectively. Overall, 97.4% and 98.6% of samples gave valid results using the cobas and HBM, respectively. Of the 185 samples strictly fulfilling the cobas guidelines, the concordance rate was even higher (95.7%; kappa = 0.91; 95%CI [0.85; 0.97]. Out of the 420 samples tested, 28 (6.7% showed discordance between HBM and cobas. This prospective study shows a high concordance rate between the cobas 4800 BRAF V600 test and home brew methods in the routine detection of BRAF V600E mutations.

BFD-22 a new potential inhibitor of BRAF inhibits the metastasis of B16F10 melanoma cells and simultaneously increased the tumor immunogenicity

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Ferreira, Adilson Kleber, E-mail: ferreira-kleber@usp.br [Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP (Brazil); Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, Groningen (Netherlands); Pasqualoto, Kerly Fernanda Mesquita [Biochemistry and Biophysics Laboratory, Butantan Institute, São Paulo, SP (Brazil); Kruyt, Frank A.E. [Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, Groningen (Netherlands); Palace-Berl, Fanny [Laboratory of Drug Design and Development, University of São Paulo, São Paulo, SP (Brazil); Azevedo, Ricardo Alexandre [Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP (Brazil); Turra, Kely Medeiros [Laboratory of Cytopathology, Department of Clinical Chemistry and Toxicology, Faculty of Pharmaceutical Sciences, University of São Paulo, São Paulo, SP (Brazil); Rodrigues, Cecilia Pessoa; Ferreira, Ana Carolina Franco; Salomón, Maria Alejandra Clavijo [Laboratory of Tumor Immunology, University of São Paulo, São Paulo, SP (Brazil); Sá, Paulo Luiz de [Laboratory of Genetics, Butantan Institute, São Paulo, SP (Brazil); Farias, Camyla Fernandes; Figueiredo, Carlos Rogerio [Experimental Oncology Section, The Federal University of São Paulo, São Paulo, SP (Brazil); and others

2016-03-15

Benzofuroxan is an interesting ring system, which has shown a wide spectrum of biological responses against tumor cell lines. We investigated, herein, the antitumor effects of benzofuroxan derivatives (BFDs) in vitro and in a melanoma mouse model. Cytotoxic effects of twenty-two BFDs were determined by MTT assay. Effects of BFD-22 in apoptosis and cell proliferation were evaluated using Annexin V-FITC/PI and CFSE staining. In addition, the effects in the cell cycle were assessed. Flow cytometry, western blot, and fluorescence microscopy analysis were employed to investigate the apoptosis-related proteins and the BRAF signaling. Cell motility was also exploited through cell invasion and migration assays. Molecular docking approach was performed in order to verify the BFD-22 binding mode into the ATP catalytic site of BRAF kinase. Moreover, the BFD-22 antitumor effects were evaluated in a melanoma murine model using B16F10. BFD-22 was identified as a potential hit against melanoma cells. BFD-22 induced apoptosis and inhibited cell proliferation of B16F10 cells. BFD-22 has suppressed, indeed, the migratory and invasive behavior of B16F10 cells. Cyclin D1 and CDK4 expression were reduced leading to cell cycle arrest at G0/G1 phase. Of note, phosphorylation of BRAF at Ser338 was strongly down-regulated by BFD-22 in B16F10 cells. The accommodation/orientation into the binding site of BRAF was similar of BAY43-9006 (co-crystallized inhibitor of BRAF, sorafenib). Importantly, BFD-22 presented in vivo antimetastatic effects and showed better therapeutic efficacy than sorafenib and taxol. BFD-22 can be considered as a new lead compound and, then, can be helpful for the designing of novel drug candidates to treat melanoma. - Highlights: • BFD-22 induces apoptosis effects of B16F10 cells by mitochondrial pathway. • BFD-22 provokes downstream in the MAPK/ERK kinase signaling cascade. • Molecular docking trials supported BRAF protein as potential target for BDF-22. • BFD

Potential relationship between Hashimoto's thyroiditis and BRAF(V600E) mutation status in papillary thyroid cancer.

Science.gov (United States)

Zeng, Rui-Chao; Jin, Lang-Ping; Chen, En-Dong; Dong, Si-Yang; Cai, Ye-Feng; Huang, Guan-Li; Li, Quan; Jin, Chun; Zhang, Xiao-Hua; Wang, Ou-Chen

2016-04-01

The purpose of this study was to evaluate the potential relationship between Hashimoto's thyroiditis and BRAF(V600E) mutation status in patients with papillary thyroid carcinoma (PTC). A total of 619 patients with PTC who underwent total thyroidectomy with lymph node dissection were enrolled in this study. Univariable and multivariate analyses were used. Hashimoto's thyroiditis was present in 35.9% (222 of 619) of PTCs. Multivariate logistic regressions showed that BRAF(V600E) mutation, sex, extrathyroidal extension, and lymph node metastasis were independent factors for Hashimoto's thyroiditis. Female sex, more frequent extrathyroidal extension, and a higher incidence of lymph node metastasis were significantly associated with PTCs accompanied by BRAF(V600E) mutation without Hashimoto's thyroiditis compared with PTCs accompanied by BRAF(V600E) mutation with Hashimoto's thyroiditis. Hashimoto's thyroiditis was negatively associated with BRAF(V600E) mutation, extrathyroidal extension, and lymph node metastasis. In addition, Hashimoto's thyroiditis was related to less lymph node metastasis and extrathyroidal extension in PTCs with BRAF(V600E) mutation. Therefore, Hashimoto's thyroiditis is a potentially protective factor in PTC. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1019-E1025, 2016. © 2015 Wiley Periodicals, Inc.

Mutational analysis of BRAF and KRAS in ovarian serous borderline (atypical proliferative) tumours and associated peritoneal implants

DEFF Research Database (Denmark)

Ardighieri, Laura; Zeppernick, Felix; Hannibal, Charlotte G

2014-01-01

There is debate as to whether peritoneal implants associated with serous borderline tumours/atypical proliferative serous tumours (SBT/APSTs) of the ovary are derived from the primary ovarian tumour or arise independently in the peritoneum. We analysed 57 SBT/APSTs from 45 patients with advanced......), 34 (53.9%) had KRAS mutations and 14 (22%) had BRAF mutations, of which identical KRAS mutations were found in 34 (91%) of 37 SBT/APST-implant pairs and identical BRAF mutations in 14 (100%) of 14 SBT/APST-implant pairs. Wild-type KRAS and BRAF (at the loci investigated) were found in 11 (100%) of 11...... SBT/APST-implant pairs. Overall concordance of KRAS and BRAF mutations was 95% in 59 of 62 SBT/APST-implant (non-invasive and invasive) pairs (p identical KRAS or BRAF...

BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma

Science.gov (United States)

Frederick, Dennie Tompers; Piris, Adriano; Cogdill, Alexandria P.; Cooper, Zachary A.; Lezcano, Cecilia; Ferrone, Cristina R.; Mitra, Devarati; Boni, Andrea; Newton, Lindsay P.; Liu, Chengwen; Peng, Weiyi; Sullivan, Ryan J; Lawrence, Donald P.; Hodi, F. Stephen; Overwijk, Willem W.; Lizée, Gregory; Murphy, George F.; Hwu, Patrick; Flaherty, Keith T.; Fisher, David E.; Wargo, Jennifer A.

2013-01-01

Purpose To evaluate the effects BRAF inhibition on the tumor microenvironment in patients with metastatic melanoma. Experimental Design Thirty-five biopsies were collected from 16 patients with metastatic melanoma pretreatment (day 0) and at 10-14 days after initiation of treatment with either BRAF inhibitor alone (vemurafenib) or BRAF + MEK inhibition (dabrafenib + trametinib), and were also taken at time of progression. Biopsies were analyzed for melanoma antigens, T cell markers, and immunomodulatory cytokines. Results Treatment with either BRAF inhibitor alone or BRAF + MEK inhibitor was associated with an increased expression of melanoma antigens and an increase in CD8+ T cell infiltrate. This was also associated with a decrease in immunosuppressive cytokines (IL-6 & IL-8) and an increase in markers of T cell cytotoxicity. Interestingly, expression of exhaustion markers TIM-3 and PD1 and the immunosuppressive ligand PDL1 were increased on treatment. A decrease in melanoma antigen expression and CD8 T cell infiltrate was noted at time of progression on BRAF inhibitor alone, and was reversed with combined BRAF and MEK inhibition. Conclusions Together, this data suggests that treatment with BRAF inhibition enhances melanoma antigen expression and facilitates T cell cytotoxicity and a more favorable tumor microenvironment, providing support for potential synergy of BRAF-targeted therapy and immunotherapy. Interestingly, markers of T cell exhaustion and the immunosuppressive ligand PDL1 are also increased with BRAF inhibition, further implying that immune checkpoint blockade may be critical in augmenting responses to BRAF-targeted therapy in patients with melanoma. PMID:23307859

Hubungan BRAF V600E dan EGFR dengan Metastasis ke Kelenjar Getah Bening pada Adenokarsinoma Kolorektal

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Fenny Ariyanni

2015-09-01

Full Text Available Colorectal adenocarcinoma is an epithelial malignant tumor with glandular differentiation. Lymph node metastasis affects the prognosis and management of colorectal carcinoma patients. In this study, association of BRAF V600E and EGFR with metastasis of the lymph nodes was investigated. This was a cross sectional study with unpaired categorical analysis of colorectal adenocarcinoma obtained from archival paraffin blocks from consecutively selected samples. The blocks were stained by BRAF V600E and EGFR antibodies at the Department of Anatomical Pathology, Faculty of Medicine, Universitas Padjadjaran/Dr. Hasan Sadikin General Hospital during the period of February to June 2014. There was no association between positive BRAF V600E immunoexpression and lymph node metastasis, p=0.269 (p>0.05, chi-square test. Similarly, there was no association between positive EGFR immunoexpression and lymph node metastasis, p=0.713 (p>0.05, chi-square test. Positive BRAF V600E immunoexpresion and positive EGFR immunoexpression also had no association with lymph node metastasis, p=0.427 (Fisher Exact test. BRAF and EGFR may play a role in the epithelial mesenchymal transition to increase cell migration and invasion. However, in colorectal adenocarcinoma, BRAF V600E and EGFR were not associated with lymph node metastasis. In conclusions, positive BRAF V600E immunoexpression and positive EGFR immunoexpression in colorectal adenocarcinoma should not be used as markers for the metastazing potentials of colorectal adenocarcinoma tumors.

Significance of the BRAF mRNA Expression Level in Papillary Thyroid Carcinoma: An Analysis of The Cancer Genome Atlas Data.

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Young Jun Chai

Full Text Available BRAFV600E is the most common mutation in papillary thyroid carcinoma (PTC, and it is associated with high-risk prognostic factors. However, the significance of the BRAF mRNA level in PTC remains unknown. We evaluated the significance of BRAF mRNA expression level by analyzing PTC data from The Cancer Genome Atlas (TCGA database.Data from 499 patients were downloaded from the TCGA database. After excluding other PTC variants, we selected 353 cases of classic PTC, including 193 cases with BRAFV600E and 160 cases with the wild-type BRAF. mRNA abundances were measured using RNA-Seq with the Expectation Maximization algorithm.The mean BRAF mRNA level was significantly higher in BRAFV600E patients than in patients with wild-type BRAF (197.6 vs. 179.3, p = 0.031. In wild-type BRAF patients, the mean BRAF mRNA level was higher in cases with a tumor > 2 cm than those with a tumor ≤ 2.0 cm (189.4 vs. 163.8, p = 0.046, and was also higher in cases with lymph node metastasis than in those without lymph node metastasis (188.5 vs. 157.9, p = 0.040. Within BRAFV600E patients, higher BRAF mRNA expression was associated with extrathyroidal extension (186.4 vs. 216.4, p = 0.001 and higher T stage (188.1 vs. 210.2, p = 0.016.A higher BRAF mRNA expression level was associated with tumor aggressiveness in classic PTC regardless of BRAF mutational status. Evaluation of BRAF mRNA level may be helpful in prognostic risk stratification of PTC.

Dabrafenib and Trametinib Treatment in Patients With Locally Advanced or Metastatic BRAF V600-Mutant Anaplastic Thyroid Cancer.

Science.gov (United States)

Subbiah, Vivek; Kreitman, Robert J; Wainberg, Zev A; Cho, Jae Yong; Schellens, Jan H M; Soria, Jean Charles; Wen, Patrick Y; Zielinski, Christoph; Cabanillas, Maria E; Urbanowitz, Gladys; Mookerjee, Bijoyesh; Wang, Dazhe; Rangwala, Fatima; Keam, Bhumsuk

2018-01-01

Purpose We report the efficacy and safety of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) combination therapy in BRAF V600E-mutated anaplastic thyroid cancer, a rare, aggressive, and highly lethal malignancy with poor patient outcomes and no systemic therapies with clinical benefit. Methods In this phase II, open-label trial, patients with predefined BRAF V600E-mutated malignancies received dabrafenib 150 mg twice daily and trametinib 2 mg once daily until unacceptable toxicity, disease progression, or death. The primary end point was investigator-assessed overall response rate. Secondary end points included duration of response, progression-free survival, overall survival, and safety. Results Sixteen patients with BRAF V600E-mutated anaplastic thyroid cancer were evaluable (median follow-up, 47 weeks; range, 4 to 120 weeks). All patients had received prior radiation treatment and/or surgery, and six had received prior systemic therapy. The confirmed overall response rate was 69% (11 of 16; 95% CI, 41% to 89%), with seven ongoing responses. Median duration of response, progression-free survival, and overall survival were not reached as a result of a lack of events, with 12-month estimates of 90%, 79%, and 80%, respectively. The safety population was composed of 100 patients who were enrolled with seven rare tumor histologies. Common adverse events were fatigue (38%), pyrexia (37%), and nausea (35%). No new safety signals were detected. Conclusion Dabrafenib plus trametinib is the first regimen demonstrated to have robust clinical activity in BRAF V600E-mutated anaplastic thyroid cancer and was well tolerated. These findings represent a meaningful therapeutic advance for this orphan disease.

Noninferior response in BRAF{sup V600E} mutant nonmetastatic papillary thyroid carcinoma to radioiodine therapy

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Li, Jiao; Zhao, Teng; Lin, Yansong [Peking Union Medical College Hospital, Department of Nuclear Medicine, Beijing (China); Liang, Jun [Peking University International Hospital, Department of Oncology, Beijing (China)

2016-06-15

As the most frequent and specific genetic alteration in papillary thyroid carcinoma (PTC), BRAF{sup V600E} has an intimate relationship with more invasive tumour and higher postoperative recurrence risk in PTC patients. We investigate the effect of radioactive iodine (RAI) therapy on the clinical outcome in PTC patients with the BRAF{sup V600E} mutation without distant metastases. This retrospective study included PTC 228 patients without distant metastases who underwent total or near-total thyroidectomy and RAI treatment in our hospital from January 2011 to July 2014. The BRAF{sup V600E} status of the primary lesions was determined and the patients were divided into two groups according to the presence of the mutation. Serological and imaging data were collected at a median follow-up of 2.34 years after RAI administration. Suppressed and stimulated thyroglobulin (Tg), Tg antibody, diagnostic whole-body scintigraphy, and other imaging examinations were used to assess clinical outcome, which was defined as excellent response, indeterminate response, biochemical incomplete response and structural incomplete response. The BRAF{sup V600E} mutation was observed in 153 of the 228 patients (67.1 %). The clinicopathological features did not differ between the BRAF{sup V600E} mutatation and wild-type groups except age at diagnosis (P = 0.000), tumour size (P = 0.023) and TNM stage (P = 0.003). Older age and more advanced TNM stage were prevalent in the BRAF{sup V600E} mutatation group, whereas tumours were slightly larger in the BRAF{sup V600E} wild-type group. The response to RAI therapy was evaluated in both the entire series and the patients with a high recurrence risk, and no significant difference in response was found between the BRAF{sup V600E} mutatation and the wild-type groups (P = 0.881 and P = 0.851, respectively). The clinical response to timely postsurgical RAI therapy is not inferior in BRAF{sup V600E} mutation PTC patients without distant metastases, which

Dabrafenib: a new opportunity for the treatment of BRAF V600-positive melanoma

Directory of Open Access Journals (Sweden)

Banzi M

2016-05-01

Full Text Available Maria Banzi,1 Simona De Blasio,2 Aimilios Lallas,3 Caterina Longo,2 Elvira Moscarella,2 Roberto Alfano,4 Giuseppe Argenziano5 1Department of Medical Oncology, Arcispedale Santa Maria Nuova IRCCS, Reggio Emilia, Italy; 2Skin Cancer Unit, Arcispedale Santa Maria Nuova IRCCS, Reggio Emilia, Italy; 3First Department of Dermatology, Aristotle University, Thessaloniki, Greece; 4Department of Anesthesiology, Surgery and Emergency, 5Dermatology Unit, Second University of Naples, Naples, Italy Abstract: Prior to 2011, the 1-year survival rates for patients suffering from advanced or metastatic melanoma was as low as 33%, with a median overall survival of about 9 months. Several chemotherapeutic regimens have been applied, either as monochemotherapy or as polychemotherapy, overall not resulting in an improvement of progression-free or overall survival. Novel insights into the epidemiology and biology of melanoma allowed the development of newer therapies. The discovery of mutations in BRAF, a part of the mitogen-activated protein kinase, allowed the development of two BRAF inhibitors, vemurafenib and dabrafenib, which significantly improved the outcome of metastatic melanoma treatment. This article reviews the mechanism of action, efficacy, and safety profile of dabrafenib. An in-depth knowledge of this medication will encourage clinicians to select the appropriate therapeutic strategy for each patient, as well as to prevent or adequately manage side effects, optimizing, thus, the drug’s applicability. Keywords: melanoma, BRAF, target therapy, dabrafenib, melanoma survival

BRAF V600E mutations are common in pleomorphic xanthoastrocytoma: diagnostic and therapeutic implications.

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Dora Dias-Santagata

2011-03-01

Full Text Available Pleomorphic xanthoastrocytoma (PXA is low-grade glial neoplasm principally affecting children and young adults. Approximately 40% of PXA are reported to recur within 10 years of primary resection. Upon recurrence, patients receive radiation therapy and conventional chemotherapeutics designed for high-grade gliomas. Genetic changes that can be targeted by selective therapeutics have not been extensively evaluated in PXA and ancillary diagnostic tests to help discriminate PXA from other pleomorphic and often more aggressive astrocytic malignancies are limited. In this study, we apply the SNaPshot multiplexed targeted sequencing platform in the analysis of brain tumors to interrogate 60 genetic loci that are frequently mutated in 15 cancer genes. In our analysis we detect BRAF V600E mutations in 12 of 20 (60% WHO grade II PXA, in 1 of 6 (17% PXA with anaplasia and in 1 glioblastoma arising in a PXA. Phospho-ERK was detected in all tumors independent of the BRAF mutation status. BRAF duplication was not detected in any of the PXA cases. BRAF V600E mutations were identified in only 2 of 71 (2.8% glioblastoma (GBM analyzed, including 1 of 9 (11.1% giant cell GBM (gcGBM. The finding that BRAF V600E mutations are common in the majority of PXA has important therapeutic implications and may help in differentiating less aggressive PXAs from lethal gcGBMs and GBMs.

Context-dependent interpretation of the prognostic value of BRAF and KRAS mutations in colorectal cancer

International Nuclear Information System (INIS)

Popovici, Vlad; Budinska, Eva; Bosman, Fred T; Tejpar, Sabine; Roth, Arnaud D; Delorenzi, Mauro

2013-01-01

The mutation status of the BRAF and KRAS genes has been proposed as prognostic biomarker in colorectal cancer. Of them, only the BRAF V600E mutation has been validated independently as prognostic for overall survival and survival after relapse, while the prognostic value of KRAS mutation is still unclear. We investigated the prognostic value of BRAF and KRAS mutations in various contexts defined by stratifications of the patient population. We retrospectively analyzed a cohort of patients with stage II and III colorectal cancer from the PETACC-3 clinical trial (N = 1,423), by assessing the prognostic value of the BRAF and KRAS mutations in subpopulations defined by all possible combinations of the following clinico-pathological variables: T stage, N stage, tumor site, tumor grade and microsatellite instability status. In each such subpopulation, the prognostic value was assessed by log rank test for three endpoints: overall survival, relapse-free survival, and survival after relapse. The significance level was set to 0.01 for Bonferroni-adjusted p-values, and a second threshold for a trend towards statistical significance was set at 0.05 for unadjusted p-values. The significance of the interactions was tested by Wald test, with significance level of 0.05. In stage II-III colorectal cancer, BRAF mutation was confirmed a marker of poor survival only in subpopulations involving microsatellite stable and left-sided tumors, with higher effects than in the whole population. There was no evidence for prognostic value in microsatellite instable or right-sided tumor groups. We found that BRAF was also prognostic for relapse-free survival in some subpopulations. We found no evidence that KRAS mutations had prognostic value, although a trend was observed in some stratifications. We also show evidence of heterogeneity in survival of patients with BRAF V600E mutation. The BRAF mutation represents an additional risk factor only in some subpopulations of colorectal cancers, in

Mutação BRAF em pacientes idosos submetidos à tireoidectomia

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Antonio Augusto T. Bertelli

Full Text Available OBJETIVO: Avaliar a frequência da mutação V600E do gene BRAF em pacientes com mais de 65 anos de idade submetidos à tireoidectomia, correlacionando sua presença ou ausência com as diferentes lesões histológicas, com as variantes e com fatores prognósticos do carcinoma papilífero. MÉTODOS: Foram avaliados 85 pacientes com mais de 65 anos de idade submetidos à tireoidectomia, analisando a mutação BRAF V600E através de reação de PCR-RT realizada após a extração do DNA dos blocos de parafina. RESULTADOS: Detectou-se ausência ou presença da mutação BRAF V600E em 47 pacientes (55,3%. Entre os 17 carcinomas papilíferos estudados, sete apresentavam a mutação (41,2%. Demonstrou-se associação estatística entre a presença desta mutação e a variante clássica do carcinoma papilífero, além de tendência de associação com o extravasamento tireoideano. CONCLUSÃO: A mutação BRAF nos pacientes idosos também é exclusiva do carcinoma papilífero e tem frequência expressiva. Além disso, está relacionada à variante clássica e, possivelmente, ao extravasamento tireoideano.

MLH1-deficient Colorectal Carcinoma With Wild-type BRAF and MLH1 Promoter Hypermethylation Harbor KRAS Mutations and Arise From Conventional Adenomas.

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Farchoukh, Lama; Kuan, Shih-Fan; Dudley, Beth; Brand, Randall; Nikiforova, Marina; Pai, Reetesh K

2016-10-01

Between 10% and 15% of colorectal carcinomas demonstrate sporadic DNA mismatch-repair protein deficiency as a result of MLH1 promoter methylation and are thought to arise from sessile serrated adenomas, termed the serrated neoplasia pathway. Although the presence of the BRAF V600E mutation is indicative of a sporadic cancer, up to 30% to 50% of colorectal carcinomas with MLH1 promoter hypermethylation will lack a BRAF mutation. We report the clinicopathologic and molecular features of MLH1-deficient colorectal carcinoma with wild-type BRAF and MLH1 promoter hypermethylation (referred to as MLH1-hypermethylated BRAF wild-type colorectal carcinoma, n=36) in comparison with MLH1-deficient BRAF-mutated colorectal carcinoma (n=113) and Lynch syndrome-associated colorectal carcinoma (n=36). KRAS mutations were identified in 31% of MLH1-hypermethylated BRAF wild-type colorectal carcinomas compared with 0% of MLH1-deficient BRAF-mutated colorectal carcinomas and 37% of Lynch syndrome-associated colorectal carcinomas. When a precursor polyp was identified, MLH1-hypermethylated BRAF wild-type colorectal carcinomas arose from precursor polyps resembling conventional tubular/tubulovillous adenomas in contrast to MLH1-deficient BRAF-mutated colorectal carcinomas, which arose from precursor sessile serrated adenomas (PMLH1-hypermethylated BRAF wild-type colorectal carcinoma and MLH1-deficient BRAF-mutated colorectal carcinoma had a predilection for the right colon compared with Lynch syndrome-associated colorectal carcinoma (86% vs. 92% vs. 49%, P0.05). In conclusion, our results indicate that MLH1-hypermethylated BRAF wild-type colorectal carcinomas can harbor KRAS mutations and arise from precursor polyps resembling conventional tubular/tubulovillous adenomas.

Systems Analysis of Adaptive Responses to MAP Kinase Pathway Blockade in BRAF Mutant Melanoma.

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Brian J Capaldo

Full Text Available Fifty percent of cutaneous melanomas are driven by activated BRAFV600E, but tumors treated with RAF inhibitors, even when they respond dramatically, rapidly adapt and develop resistance. Thus, there is a pressing need to identify the major mechanisms of intrinsic and adaptive resistance and develop drug combinations that target these resistance mechanisms. In a combinatorial drug screen on a panel of 12 treatment-naïve BRAFV600E mutant melanoma cell lines of varying levels of resistance to mitogen-activated protein kinase (MAPK pathway inhibition, we identified the combination of PLX4720, a targeted inhibitor of mutated BRaf, and lapatinib, an inhibitor of the ErbB family of receptor tyrosine kinases, as synergistically cytotoxic in the subset of cell lines that displayed the most resistance to PLX4720. To identify potential mechanisms of resistance to PLX4720 treatment and synergy with lapatinib treatment, we performed a multi-platform functional genomics analysis to profile the genome as well as the transcriptional and proteomic responses of these cell lines to treatment with PLX4720. We found modest levels of resistance correlated with the zygosity of the BRAF V600E allele and receptor tyrosine kinase (RTK mutational status. Layered over base-line resistance was substantial upregulation of many ErbB pathway genes in response to BRaf inhibition, thus generating the vulnerability to combination with lapatinib. The transcriptional responses of ErbB pathway genes are associated with a number of transcription factors, including ETS2 and its associated cofactors that represent a convergent regulatory mechanism conferring synergistic drug susceptibility in the context of diverse mutational landscapes.

Dabrafenib and Trametinib Treatment in Patients With Locally Advanced or Metastatic BRAF V600–Mutant Anaplastic Thyroid Cancer

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Subbiah, Vivek; Kreitman, Robert J.; Wainberg, Zev A.; Cho, Jae Yong; Schellens, Jan H.M.; Soria, Jean Charles; Wen, Patrick Y.; Zielinski, Christoph; Cabanillas, Maria E.; Urbanowitz, Gladys; Mookerjee, Bijoyesh; Wang, Dazhe; Rangwala, Fatima

2018-01-01

Purpose We report the efficacy and safety of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) combination therapy in BRAF V600E–mutated anaplastic thyroid cancer, a rare, aggressive, and highly lethal malignancy with poor patient outcomes and no systemic therapies with clinical benefit. Methods In this phase II, open-label trial, patients with predefined BRAF V600E–mutated malignancies received dabrafenib 150 mg twice daily and trametinib 2 mg once daily until unacceptable toxicity, disease progression, or death. The primary end point was investigator-assessed overall response rate. Secondary end points included duration of response, progression-free survival, overall survival, and safety. Results Sixteen patients with BRAF V600E–mutated anaplastic thyroid cancer were evaluable (median follow-up, 47 weeks; range, 4 to 120 weeks). All patients had received prior radiation treatment and/or surgery, and six had received prior systemic therapy. The confirmed overall response rate was 69% (11 of 16; 95% CI, 41% to 89%), with seven ongoing responses. Median duration of response, progression-free survival, and overall survival were not reached as a result of a lack of events, with 12-month estimates of 90%, 79%, and 80%, respectively. The safety population was composed of 100 patients who were enrolled with seven rare tumor histologies. Common adverse events were fatigue (38%), pyrexia (37%), and nausea (35%). No new safety signals were detected. Conclusion Dabrafenib plus trametinib is the first regimen demonstrated to have robust clinical activity in BRAF V600E–mutated anaplastic thyroid cancer and was well tolerated. These findings represent a meaningful therapeutic advance for this orphan disease. PMID:29072975

Critical Pitfalls in the use of BRAF Mutation as a Diagnostic Tool in Thyroid Nodules: a Case Report.

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Kuhn, Elisabetta; Ragazzi, Moira; Zini, Michele; Giordano, Davide; Nicoli, Davide; Piana, Simonetta

2016-09-01

Thyroid fine-needle aspiration (FNA) cytology is the primary tool for the diagnostic evaluation of thyroid nodules. BRAF mutation analysis is employed as an ancillary tool in indeterminate cases, as recommended by the American Thyroid Association management guidelines. Hereby, we report the case of a 73-year-old woman who presented an 8-mm-size, ill-defined, left thyroid nodule. FNA resulted "suspicious for papillary thyroid carcinoma". BRAF mutation status was analyzed, and somatic BRAF (V600E) mutation identified. The patient underwent a total thyroidectomy. At histological examination, the nodule was composed of Langerhans cells, admixed with many eosinophils. A final diagnosis of Langerhans cell histiocytosis of the thyroid was made. Our case emphasizes the critical diagnostic pitfalls due to the use of BRAF (V600E) mutation analysis in thyroid FNA. Notably, BRAF (V600E) mutation is common in melanoma, colorectal carcinoma, lung carcinoma, ovarian carcinoma, brain tumors, hairy cell leukemia, multiple myeloma, and histiocytoses. Therefore, in cases of indeterminate FNA with unclassifiable atypical cells BRAF (V600E) mutated, the possibility of a localization of hystiocytosis or a secondary thyroid malignancy should be taken into account.

Quantitative analysis of wild-type and V600E mutant BRAF proteins in colorectal carcinoma using immunoenrichment and targeted mass spectrometry

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Chen, Hang [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang 110122 (China); Hsiao, Yung-Chin [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Chiang, Sum-Fu [Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Graduate Institute of Clinical Medical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Wu, Chia-Chun; Lin, Yu-Tsun [Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Liu, Hsuan [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Zhao, Hong [Experimental Center of Functional Subjects, College of Basic Medicine, China Medical University, Shenyang, Liaoning 110122 (China); Chen, Jinn-Shiun [Division of Colon and Rectal Surgery, Chang Gung Memorial Hospital, Linkou, Taiwan (China); Chang, Yu-Sun [Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan (China); Department of Otolaryngology, Chang Gung Memorial Hospital, Linkou, Taiwan (China); and others

2016-08-24

The BRAF V600E mutation is one of the most common mutations implicated in the development of several types of cancer including colorectal cancer (CRC), where it is associated with aggressive disease phenotypes and poor outcomes. The status of the BRAF V600E mutation is frequently determined by direct DNA sequencing. However, no previous study has sought to quantify the BRAF V600E protein in cancer specimens. Here, we evaluated immunoenrichment coupled with two MS-based quantitative techniques, namely multiple reaction monitoring (MRM) and single ion monitoring conjugated accurate inclusion mass screening (SIM-AIMS), to detect and precisely quantify wild-type (WT) and V600E mutant BRAF proteins in DNA sequence-confirmed CRC tissue specimens. WT and V600E BRAF proteins were immunoprecipitated from a CRC cell line (HT-29), and their representative peptides ({sup 592}IGDFGLATVK{sup 601} and {sup 592}IGDFGLATEK{sup 601}, respectively) were confirmed by LC-MS/MS analysis and then quantified by MRM or SIM-AIMS with spiked stable isotope-labeled peptide standards. Both assays worked well for measuring WT BRAF from different amounts of HT-29 cell lysates, but the MRM assay was more sensitive than SIM-AIMS assay for quantifying lower levels of V600E BRAF. In protein extracts (2 mg) from 11 CRC tissue specimens, the MRM assay could measure WT BRAF in all 11 cases (0.32–1.66 ng) and the V600E BRAF in two cases (0.1–0.13 ng; mutant-to-WT ratio, 0.16–0.17). The SIM-AIMS assay could also detect WT and V600E BRAF in CRC specimens, but the measured levels of both targets were lower than those determined by MRM assay. Collectively, this study provides an effective method to precisely quantify WT and V600E BRAF proteins in complex biological samples using immunoenrichment-coupled targeted MS. Since the V600E BRAF protein has emerged as an important therapeutic target for cancer, the developed assay should facilitate future BRAF-related basic and clinical studies

Impact of KRAS, BRAF and PI3KCA mutations in rectal carcinomas treated with neoadjuvant radiochemotherapy and surgery

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Derbel, Olfa; La Fouchardière, Christelle de; Wang, Qing; Desseigne, Françoise; Rivoire, Michel; Meeus, Pierre; Peyrat, Patrice; Stella, Mattia; Martel-Lafay, Isabelle; Lemaistre, Anne-Isabelle

2013-01-01

Conventional treatment for locally advanced rectal cancer usually combines neoadjuvant radiochemotherapy and surgery. Until recently, there have been limited predictive factors (clinical or biological) for rectal tumor response to conventional treatment. KRAS, BRAF and PIK3CA mutations are commonly found in colon cancers. In this study, we aimed to determine the mutation frequencies of KRAS, BRAF and PIK3CA and to establish whether such mutations may be used as prognostic and/or predictive factors in rectal cancer patients. We retrospectively reviewed the clinical and biological data of 98 consecutive operated patients between May 2006 and September 2009. We focused in patients who received surgery in our center after radiochemotherapy and in which tumor samples were available. In the 98 patients with a rectal cancer, the median follow-up time was 28.3 months (4–74). Eight out of ninety-eight patients experienced a local recurrence (8%) and 17/98 developed distant metastasis (17%). KRAS, BRAF and PIK3CA were identified respectively in 23 (23.5%), 2 (2%) and 4 (4%) patients. As described in previous studies, mutations in KRAS and BRAF were mutually exclusive. No patient with local recurrence exhibited KRAS or PIK3CA mutation and one harbored BRAF mutation (12.5%). Of the seventeen patients with distant metastasis (17%), 5 were presenting KRAS mutation (29%), one BRAF (5%) and one PIK3CA mutation (5%). No relationship was seen between PIK3CA, KRAS or BRAF mutation and local or distant recurrences. The frequencies of KRAS, BRAF and PIK3CA mutations in our study were lower than the average frequencies reported in colorectal cancers and no significant correlation was found between local/distant recurrences and KRAS, BRAF or PIK3CA mutations. Future studies with greater number of patients, longer follow-up time and greater power to predict associations are necessary to fully understand this relationship

Fisetin, a phytochemical, potentiates sorafenib-induced apoptosis and abrogates tumor growth in athymic nude mice implanted with BRAF-mutated melanoma cells.

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Pal, Harish Chandra; Baxter, Ronald D; Hunt, Katherine M; Agarwal, Jyoti; Elmets, Craig A; Athar, Mohammad; Afaq, Farrukh

2015-09-29

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.

BAG3 down-modulation reduces anaplastic thyroid tumor growth by enhancing proteasome-mediated degradation of BRAF protein.

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Chiappetta, Gennaro; Basile, Anna; Arra, Claudio; Califano, Daniela; Pasquinelli, Rosa; Barbieri, Antonio; De Simone, Veronica; Rea, Domenica; Giudice, Aldo; Pezzullo, Luciano; De Laurenzi, Vincenzo; Botti, Gerardo; Losito, Simona; Conforti, Daniela; Turco, Maria Caterina

2012-01-01

Anaplastic thyroid tumors (ATC) express high levels of BAG3, a member of the BAG family of cochaperone proteins that is involved in regulating cell apoptosis through multiple mechanisms. The objective of the study was the investigation of the influence of B-cell lymphoma-2-associated athanogene 3 (BAG3) on ATC growth. We investigated the effects of BAG3 down-modulation, obtained by using a specific small interfering RNA, on in vitro and in vivo growth of the human ATC cell line 8505C. Because BRAF protein plays an important role in ATC cell growth, we analyzed the effects of BAG3 down-modulation on BRAF protein levels. Furthermore, by using a proteasome inhibitor, we verified whether BAG3-mediated regulation of BRAF levels involved a proteasome-dependent mechanism. BAG3 down-modulation significantly inhibits ATC growth in vitro and in vivo. BAG3 coimmunoprecipitates with BRAF protein, and its down-modulation results in a significant reduction of BRAF protein levels, which can be reverted by incubation with the proteasome inhibitor MG132. BAG3 protein sustains ATC growth in vitro and in vivo. The underlying molecular mechanism appears to rely on BAG3 binding to BRAF, thus protecting it from proteasome-dependent degradation. These results are in line with the reported ability of BAG3 to interfere with the proteasomal delivery of a number of other client proteins.

Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules

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Rowe Leslie R

2006-04-01

Full Text Available Abstract Background Fine needle aspiration (FNA is widely utilized for evaluation of patients with thyroid nodules. However, approximately 30% are indeterminate for malignancy. Recently, a mutation in the BRAF gene has been reported to be the most common genetic event in papillary thyroid carcinoma (PTC. In this retrospective study, we assessed the utility of BRAF V600E mutation detection for refining indeterminate preoperative cytologic diagnoses in patients with PTC. Methods Archival indeterminate thyroid FNAs and corresponding formalin-fixed, paraffin-embedded (FFPE surgical samples with PTC were identified in our patient files. DNA extracted from slide scape lysates and 5 μm FFPE sections were evaluated for the BRAF V600E mutation using LightCycler PCR and fluorescent melting curve analysis (LCPCR. Amplification products that showed deviation from the wild-type genomic DNA melting peak, discordant FNA and FFPE matched pairs, and all benign control samples, underwent direct DNA sequencing. Results A total of 19 indeterminate thyroid FNAs demonstrating PTC on FFPE surgical samples were included in the study. Using BRAF mutation analysis, the preoperative diagnosis of PTC was confirmed in 3/19 (15.8% FNA samples that could not be conclusively diagnosed on cytology alone. However, 9/19 (47.4% FFPE tissue samples were positive for the V600E mutation. Of the discordant pairs, 5/6 FNAs contained less than 50% tumor cells. Conclusion When used with indeterminate FNA samples, BRAF mutation analysis may be a useful adjunct technique for confirming the diagnosis of malignancy in an otherwise equivocal case. However, overall tumor cell content of some archival FNA smear slides is a limiting factor for mutation detection.

First-Line Use of Vemurafenib to Enable Thyroidectomy and Radioactive Iodine Ablation for BRAF-Positive Metastatic Papillary Thyroid Carcinoma

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Bao D. Dao MD

2015-09-01

Full Text Available Background. Patients with metastatic or radioactive iodine refractory papillary thyroid carcinoma (PTC have poor prognosis due to ineffective therapy for this condition beyond surgery and radioactive iodine (RAI or 131I. BRAF mutation occurs in more than 44% of PCT. Tyrosine kinase inhibitors, the most commonly used agents for these patients, have weak BRAF inhibition activity. BRAF inhibitors have demonstrated promising efficacy in relapsed metastatic PCT after standard treatment, though they are not currently approved for this indication. Case Presentation. We present the case of a 48-year-old Hispanic male who initially presented with columnar-cell variant subtype of PTC and positive BRAFV600E mutation. The patient had widespread bulky metastases to lungs, chest wall, brain, and bone. Discussion. Initial use of vemurafenib demonstrated a 42% cytoreduction of targeted pulmonary metastases and facilitated thyroidectomy and RAI treatment. The patient achieved a durable response over 21 months in the setting of widely metastatic disease. Conclusion. Vemurafenib may be effectively used for cytoreduction in patients with bulky metastatic PTC to bridge them to thyroidectomy and RAI treatment.

KRAS and BRAF Mutations and PTEN Expression Do Not Predict Efficacy of Cetuximab-Based Chemoradiotherapy in Locally Advanced Rectal Cancer

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Erben, Philipp, E-mail: philipp.erben@medma.uni-heidelberg.de [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Stroebel, Philipp [Pathologisches Institut, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Horisberger, Karoline [Chirurgische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Popa, Juliana; Bohn, Beatrice; Hanfstein, Benjamin [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Kaehler, Georg; Kienle, Peter; Post, Stefan [Chirurgische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Wenz, Frederik [Klinik fuer Strahlentherapie und Radioonkologie, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Hochhaus, Andreas [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Klinik fuer Innere Medizin II, Abteilung Haematologie/Onkologie, Universitaetsklinikum Jena, Jena (Germany); Hofheinz, Ralf-Dieter [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany)

2011-11-15

Purpose: Mutations in KRAS and BRAF genes as well as the loss of expression of phosphatase and tensin homolog (PTEN) (deleted on chromosome 10) are associated with impaired activity of antibodies directed against epidermal growth factor receptor in patients with metastatic colorectal cancer. The predictive and prognostic value of the KRAS and BRAF point mutations as well as PTEN expression in patients with locally advanced rectal cancer (LARC) treated with cetuximab-based neoadjuvant chemoradiotherapy is unknown. Methods and Materials: We have conducted phase I and II trials of the combination of weekly administration of cetuximab and irinotecan and daily doses of capecitabine in conjunction with radiotherapy (45 Gy plus 5.4 Gy) in patients with LARC (stage uT3/4 or uN+). The status of KRAS and BRAF mutations was determined with direct sequencing, and PTEN expression status was determined with immunohistochemistry testing of diagnostic tumor biopsies. Tumor regression was evaluated by using standardized regression grading, and disease-free survival (DFS) was calculated according to the Kaplan-Meier method. Results: A total of 57 patients were available for analyses. A total of 31.6% of patients carried mutations in the KRAS genes. No BRAF mutations were found, while the loss of PTEN expression was observed in 9.6% of patients. Six patients achieved complete remission, and the 3-year DFS rate was 73%. No correlation was seen between tumor regression or DFS rate and a single marker or a combination of all markers. Conclusions: In the present series, no BRAF mutation was detected. The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based chemoradiotherapy and 3-year DFS.

KRAS and BRAF Mutations and PTEN Expression Do Not Predict Efficacy of Cetuximab-Based Chemoradiotherapy in Locally Advanced Rectal Cancer

International Nuclear Information System (INIS)

Erben, Philipp; Ströbel, Philipp; Horisberger, Karoline; Popa, Juliana; Bohn, Beatrice; Hanfstein, Benjamin; Kähler, Georg; Kienle, Peter; Post, Stefan; Wenz, Frederik; Hochhaus, Andreas; Hofheinz, Ralf-Dieter

2011-01-01

Purpose: Mutations in KRAS and BRAF genes as well as the loss of expression of phosphatase and tensin homolog (PTEN) (deleted on chromosome 10) are associated with impaired activity of antibodies directed against epidermal growth factor receptor in patients with metastatic colorectal cancer. The predictive and prognostic value of the KRAS and BRAF point mutations as well as PTEN expression in patients with locally advanced rectal cancer (LARC) treated with cetuximab-based neoadjuvant chemoradiotherapy is unknown. Methods and Materials: We have conducted phase I and II trials of the combination of weekly administration of cetuximab and irinotecan and daily doses of capecitabine in conjunction with radiotherapy (45 Gy plus 5.4 Gy) in patients with LARC (stage uT3/4 or uN+). The status of KRAS and BRAF mutations was determined with direct sequencing, and PTEN expression status was determined with immunohistochemistry testing of diagnostic tumor biopsies. Tumor regression was evaluated by using standardized regression grading, and disease-free survival (DFS) was calculated according to the Kaplan–Meier method. Results: A total of 57 patients were available for analyses. A total of 31.6% of patients carried mutations in the KRAS genes. No BRAF mutations were found, while the loss of PTEN expression was observed in 9.6% of patients. Six patients achieved complete remission, and the 3-year DFS rate was 73%. No correlation was seen between tumor regression or DFS rate and a single marker or a combination of all markers. Conclusions: In the present series, no BRAF mutation was detected. The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based chemoradiotherapy and 3-year DFS.

KRAS and BRAF mutations in anal carcinoma

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Serup-Hansen, Eva; Linnemann, Dorte; Høgdall, Estrid

2015-01-01

the frequency and the prognostic value of KRAS and BRAF mutations in a large cohort of patients with anal cancer. One hundred and ninety-three patients with T1-4N0-3M0-1 anal carcinoma were included in the study. Patients were treated with curative (92%) or palliative intent (8%) between January 2000...

Combined effect of Hashimoto's thyroiditis and BRAF(V600E) mutation status on aggressiveness in papillary thyroid cancer.

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Kim, Su-jin; Myong, Jun Pyo; Jee, Hyeon-Gun; Chai, Young Jun; Choi, June Young; Min, Hye Sook; Lee, Kyu Eun; Youn, Yeo-Kyu

2016-01-01

The purpose of this study was to evaluate the association between Hashimoto's thyroiditis and BRAF(V600E) mutation status in patients with papillary thyroid cancer (PTC) and to determine their combined association with tumor aggressiveness in PTC. A total of 1780 patients with PTC who underwent surgery were enrolled in this study. Simple and multiple analyses were performed to determine the association between Hashimoto's thyroiditis and the BRAF(V600E) mutation in PTC. Hashimoto's thyroiditis was present in 11.5% of patients (204/1780) with PTC. Multiple logistic regressions showed that BRAF(V600E) (odds ratio [OR] = 0.493; 95% confidence interval [CI] = 0.360-0.678) and the female sex (OR = 7.146; 95% CI = 3.408-18.347) were independent factors associated with Hashimoto's thyroiditis in PTC. BRAF(V600E) mutation and the Hashimoto's thyroiditis-negative PTC group were associated with aggressive disease (OR = 3.069; 95% CI = 1.654-5.916). Hashimoto's thyroiditis was associated less frequently with BRAF(V600E) , and frequently with the female sex in patients with PTC. Hashimoto's thyroiditis and BRAF(V600E) status may help to predict clinical outcome of PTC. © 2015 Wiley Periodicals, Inc.

BRAF, KIT, NRAS, GNAQ and GNA11 mutation analysis in cutaneous melanomas in Turkish population

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Ismail Yilmaz

2015-01-01

Full Text Available Background: KIT and mitogen-activated protein kinase cascade are important for melanomagenesis. In the present study, we analyzed the frequency of BRAF, NRAS, KIT, GNAQ and GNA11 gene mutations and investigated their association with clinicopathological features of melanomas in Turkish population. Materials and Methods: Forty-seven primary cutaneous melanomas were included in our study. Sanger sequencing method was used for mutation analysis in all cases. Results: Mean age was 62.1 (29-101 years. Female:male ratio was 17:30. Among 47 melanomas, 14 (29.8% BRAF, 10 (21.3% NRAS, 4 (8.5% KIT and 1(2.1% GNAQ gene mutations were detected. Two of the KIT mutations were found in acral lentiginous melanoma (ALM. In the head and neck region, mutation frequency was significantly lower than in other locations (P = 0.035. The only GNAQ gene mutation (p.Q209L was detected in a melanoma arising from blue nevus located on the scalp. None of the melanomas harbored NRAS exon 2, KIT exon 13/17/18, GNAQ exon 4 and GNA11 exon 4/5 mutations. Overall mutation frequency did not show significant difference between metastatic (8/14, 57.1% and nonmetastatic (18/33, 54.5% patients. We did not observe any significant association between mutation status and gender or age of various patients. Conclusions: Our results support that BRAF and NRAS gene mutations are common in cutaneous melanomas. The activating mutations of KIT gene are rare and especially seen in ALM. GNAQ and GNA11 mutations are infrequent in cutaneous melanomas and may be associated only with melanomas arising from blue nevus.

Melanoma cells show a heterogeneous range of sensitivity to ionizing radiation and are radiosensitized by inhibition of B-RAF with PLX-4032

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Sambade, Maria J.; Peters, Eldon C.; Thomas, Nancy E.; Kaufmann, William K.; Kimple, Randall J.; Shields, Janiel M.

2011-01-01

Purpose: To assess the relative radiosensitivities of a large collection of melanoma cell lines and to determine whether pharmacologic inhibition of mutant B-RAF with PLX-4032 can radiosensitize B-Raf+ melanoma cells. Materials and methods: A large collection of melanoma cell lines (n = 37) were treated with 0-8 Gy IR and clonogenic survival assays used to generate survival curves to rank relative radiosensitivities among the cell lines. The ability of a B-RAF inhibitor, PLX-4032, to radiosensitize highly radioresistant B-Raf+ cells was also assessed by clonogenic cell survival and spheroid invasion assays and the effects of treatment on the cell cycle assessed by FACS. Results: Melanoma cell lines displayed a very large, heterogeneous range of SF2 values (1.002-0.053) with a mean of 0.51. Cell lines with surviving fractions of 0.29 or less at SF2 and SF4 were observed at a high frequency of 18.9% and 70.2%, respectively. Treatment of B-Raf+ cells with the B-RAF inhibitor PLX-4032 in combination with radiation provided enhanced inhibition of both colony formation and invasion, and radiosensitized cells through an increase in G 1 arrest. Conclusions: Our data suggest that melanomas are not uniformly radioresistant with a significant subset displaying inherent radiosensitivity. Pharmacologic inhibition of B-RAF with PLX-4032 effectively radiosensitized B-Raf+ melanoma cells suggesting that this combination approach could provide improved radiotherapeutic response in B-Raf+ melanoma patients.

p53 constrains progression to anaplastic thyroid carcinoma in a Braf-mutant mouse model of papillary thyroid cancer

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McFadden, David G.; Vernon, Amanda; Santiago, Philip M.; Martinez-McFaline, Raul; Bhutkar, Arjun; Crowley, Denise M.; McMahon, Martin; Sadow, Peter M.; Jacks, Tyler

2014-01-01

Anaplastic thyroid carcinoma (ATC) has among the worst prognoses of any solid malignancy. The low incidence of the disease has in part precluded systematic clinical trials and tissue collection, and there has been little progress in developing effective therapies. v-raf murine sarcoma viral oncogene homolog B (BRAF) and tumor protein p53 (TP53) mutations cooccur in a high proportion of ATCs, particularly those associated with a precursor papillary thyroid carcinoma (PTC). To develop an adult-onset model of BRAF-mutant ATC, we generated a thyroid-specific CreER transgenic mouse. We used a Cre-regulated BrafV600E mouse and a conditional Trp53 allelic series to demonstrate that p53 constrains progression from PTC to ATC. Gene expression and immunohistochemical analyses of murine tumors identified the cardinal features of human ATC including loss of differentiation, local invasion, distant metastasis, and rapid lethality. We used small-animal ultrasound imaging to monitor autochthonous tumors and showed that treatment with the selective BRAF inhibitor PLX4720 improved survival but did not lead to tumor regression or suppress signaling through the MAPK pathway. The combination of PLX4720 and the mapk/Erk kinase (MEK) inhibitor PD0325901 more completely suppressed MAPK pathway activation in mouse and human ATC cell lines and improved the structural response and survival of ATC-bearing animals. This model expands the limited repertoire of autochthonous models of clinically aggressive thyroid cancer, and these data suggest that small-molecule MAPK pathway inhibitors hold clinical promise in the treatment of advanced thyroid carcinoma. PMID:24711431

Evaluation of EGFR, KRAS and BRAF gene mutations in renal cell carcinoma

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Omer Bayrak

2014-08-01

Full Text Available A subset of renal cell carcinoma (RCC patients has been shown to respond to anti-EGFR therapy. As KRAS and BRAF mutations are associated with poor response to anti-EGFR therapy in some cancers, it has been suggested that screening for KRAS and BRAF mutations in RCC may be a promising strategy to identify patients who might respond to EGFR-targeted therapy. The aim of this study was to investigate the mutation status of EGFR, KRAS and BRAF in RCC patients. Renal tumors and normal renal samples from forty-eight patients who underwent radical or partial nephrectomy for kidney cancer were used in this study. Histological classification of the tumors was performed according to International Union against Cancer (UICC / American Joint Committee on Cancer (AJCC classification. Seventeen patients (48% had clear-cell RCC, 7 (20% had chromophobe RCC, and 11 patients (32% had papillary RCC. DNA isolated from the samples was subjected to melting curve mutation analysis for EGFR, BRAF and KRAS using ABI-3130 DNA sequencer. DNA sequencing analysis of RCC samples, when compared with morphologically normal matched regions, did not show any exon mutations. Our results do not support the notion that EGFR, KRAS and BRAF might be mutated in RCC. Normal 0 false false false TR X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:TR; mso-fareast-language:EN-US;}

Tumorigenesis of Papillary Thyroid Cancer Is Not BRAF-Dependent in Patients with Acromegaly

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Kim, Hee Kyung; Lee, Ji Shin; Park, Min Ho; Cho, Jin Seong; Yoon, Jee Hee; Kim, Soo Jeong; Kang, Ho-Cheol

2014-01-01

Introduction Several studies have reported a high frequency of papillary thyroid cancer (PTC) in patients with acromegaly. The aim of this study was to determine the prevalence and predictors of thyroid cancer in patients with acromegaly and to investigate the frequency of the BRAF V600E mutation in PTC patients with and without acromegaly. Materials and Methods We conducted a retrospective study of 60 patients with acromegaly. Thyroid ultrasonography (US) and US-guided fine needle aspiration were performed on nodules with sonographic features of malignancy. We selected 16 patients with non-acromegalic PTC as a control group. The BRAF V600E mutation was analyzed in paraffin-embedded surgical specimens of PTC by real-time polymerase chain reaction, and tumor specimens from patients with PTC were stained immunohistochemically with an antibody against insulin-like growth factor-1 receptor β (IGF-1Rβ). Results Thyroid cancer was found in 15 (25.0%) patients. No differences in age, sex, initial growth hormone (GH) and IGF-1 percentage of the upper limit of normal values or treatment modalities were observed between patients with and without PTC. Acromegaly was active in 12 of 15 patients at the time of PTC diagnosis; uncontrolled acromegaly had a significantly higher frequency in the PTC group (60%) than in the non-PTC group (28.9%) (p = 0.030). The BRAF V600E mutation was present in only 9.1% (1/11) of PTC patients with acromegaly, although 62.5% (10/16) of control patients with PTC had the mutation (p = 0.007). IGF-1Rβ immunostaining showed moderate-to-strong staining in all malignant PTC cells in patients with and without acromegaly. Significantly less staining for IGF-1Rβ was observed in normal adjacent thyroid tissues of PTC patients with acromegaly compared with those without (p = 0.014). Conclusion The prevalence of PTC in acromegalic patients was high (25%). An uncontrolled hyperactive GH-IGF-1 axis may play a dominant role in the development of

Value of TIRADS, BSRTC and FNA-BRAF V600E mutation analysis in differentiating high-risk thyroid nodules.

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Zhang, Yu-zhi; Xu, Ting; Cui, Dai; Li, Xiao; Yao, Qing; Gong, Hai-yan; Liu, Xiao-yun; Chen, Huan-huan; Jiang, Lin; Ye, Xin-hua; Zhang, Zhi-hong; Shen, Mei-ping; Duan, Yu; Yang, Tao; Wu, Xiao-hong

2015-11-24

The thyroid imaging reporting and data system (TIRADS) and Bethesda system for reporting thyroid cytopathology (BSRTC) have been used for interpretation of ultrasound and fine-needle aspiration cytology (FNAC) results of thyroid nodules. BRAF(V600E) mutation analysis is a molecular tool in diagnosing thyroid carcinoma. Our objective was to compare the diagnostic value of these methods in differentiating high-risk thyroid nodules. Total 220 patients with high-risk thyroid nodules were recruited in this prospective study. They all underwent ultrasound, FNAC and BRAF(V600E) mutation analysis. The sensitivity and specificity of TIRADS were 73.1% and 88.4%. BSRTC had higher specificity (97.7%) and similar sensitivity (77.6%) compared with TIRADS. The sensitivity and specificity of BRAF(V600E) mutation (85.1%, 100%) were the highest. The combination of BSRTC and BRAF(V600E) mutation analysis significantly increased the efficiency, with 97.8% sensitivity, 97.7% specificity. In patients with BSRTC I-III, the mutation rate of BRAF(V600E) was 64.5% in nodules with TIRADS 4B compared with 8.4% in nodules with TIRADS 3 or 4A (P value in differentiating high-risk thyroid nodules. The TIRADS is useful in selecting high-risk patients for FNAB and patients with BSRTC I-III for BRAF(V600E) mutation analysis.

p21-activated Kinase1(PAK1) can promote ERK activation in a kinase independent manner

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Wang, Zhipeng; Fu, Meng; Wang, Lifeng

2013-01-01

204) although phosphorylation of b-Raf (Ser445) and c-Raf (Ser 338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1 and Mek1, independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate...... MEK activity in a kinase independent manner, probably by serving as a scaffold to facilitate interaction of c-Raf....

MicroRNA-31 expression in relation to BRAF mutation, CpG island methylation and colorectal continuum in serrated lesions.

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Ito, Miki; Mitsuhashi, Kei; Igarashi, Hisayoshi; Nosho, Katsuhiko; Naito, Takafumi; Yoshii, Shinji; Takahashi, Hiroaki; Fujita, Masahiro; Sukawa, Yasutaka; Yamamoto, Eiichiro; Takahashi, Taiga; Adachi, Yasushi; Nojima, Masanori; Sasaki, Yasushi; Tokino, Takashi; Baba, Yoshifumi; Maruyama, Reo; Suzuki, Hiromu; Imai, Kohzoh; Yamamoto, Hiroyuki; Shinomura, Yasuhisa

2014-12-01

The CpG island methylator phenotype (CIMP) is a distinct form of epigenomic instability. Many CIMP-high colorectal cancers (CRCs) with BRAF mutation are considered to arise from serrated pathway. We recently reported that microRNA-31 (miR-31) is associated with BRAF mutation in colorectal tumors. Emerging new approaches have revealed gradual changes in BRAF mutation and CIMP-high throughout the colorectum in CRCs. Here, we attempted to identify a possible association between miR-31 and epigenetic features in serrated pathway, and hypothesized that miR-31 supports the "colorectal continuum" concept. We evaluated miR-31 expression, BRAF mutation and epigenetic features including CIMP status in 381 serrated lesions and 222 non-serrated adenomas and examined associations between them and tumor location (rectum; sigmoid, descending, transverse and ascending colon and cecum). A significant association was observed between high miR-31 expression and CIMP-high status in serrated lesions with BRAF mutation (p = 0.0001). In contrast, miR-31 was slightly but insignificantly associated with CIMP status in the cases with wild-type BRAF. miR-31 expression in sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that in SSAs, whereas, no significant difference was observed between traditional serrated adenomas (TSAs) and TSAs with high-grade dysplasia. The frequency of miR-31, BRAF mutation CIMP-high and MLH1 methylation increased gradually from the rectum to cecum in serrated lesions. In conclusion, miR-31 expression was associated with CIMP-high status in serrated lesions with BRAF mutation. Our data also suggested that miR-31 plays an important role in SSA evolution and may be a molecule supporting the colorectal continuum. © 2014 UICC.

BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

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Schmitt Fernando

2008-09-01

Full Text Available Abstract Background BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC. In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP, MLH1 methylation and microsatellite instability (MSI. We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied. Methods Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies. Results KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112, but statistically different from what is found in microsatellite stable (MSS tumours (P = 0.0191. Conclusion Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal

BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

International Nuclear Information System (INIS)

Velho, Sérgia; Moutinho, Cátia; Cirnes, Luís; Albuquerque, Cristina; Hamelin, Richard; Schmitt, Fernando; Carneiro, Fátima; Oliveira, Carla; Seruca, Raquel

2008-01-01

BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC). In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP), MLH1 methylation and microsatellite instability (MSI). We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied. Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies. KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112), but statistically different from what is found in microsatellite stable (MSS) tumours (P = 0.0191). Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal serrated polyps and verified that colorectal serrated

Identification of coexistence of BRAF V600E mutation and EZH2 gain specifically in melanoma as a promising target for combination therapy.

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Yu, Huan; Ma, Meng; Yan, Junya; Xu, Longwen; Yu, Jiayi; Dai, Jie; Xu, Tianxiao; Tang, Huan; Wu, Xiaowen; Li, Siming; Lian, Bin; Mao, Lili; Chi, Zhihong; Cui, Chuanliang; Guo, Jun; Kong, Yan

2017-12-04

Coexistence of enhancer of zeste homolog 2 (EZH2) and BRAF gene aberrations has been described in many cancer types. In this study, we aim to explore the coexistence status of BRAF V600E mutation and the copy number variation of EZH2 and explore the potential of this combination as a therapeutic target. A total of 138 cases of melanoma samples harboring BRAF V600E mutation were included, and EZH2 copy numbers were examined by QuantiGenePlex DNA Assays. Clinical pathological distinction between patient groups with or without EZH2 amplification (hereafter referred to as EZH2 gain) was statistically analyzed. The sensitivity of melanoma cell lines and patient-derived xenograft (PDX) models containing BRAF V600E mutation with or without EZH2 gain to vemurafenib (BRAF inhibitor), GSK2816126 (EZH2 inhibitor) and a combination of both agents was evaluated. In our cohort, the coexistence rate of BRAF V600E mutation and EZH2 gain was up to 29.0%, and significant differences in overall survival and disease-free survival were found between no EZH2 copy number gain and gain groups (P = 0.038, P = 0.030), gain and high EZH2 copy number gain groups (P = 0.006, P = 0.010). Combination with BRAF and EZH2 inhibition showed better inhibitory efficacy in melanoma prevention compared with vemurafenib monotherapy. More importantly, this improved therapeutic effect was observed especially in melanoma cell lines and PDX models containing concurrently BRAF V600E mutation and EZH2 gain. Coexistence of BRAF V600E mutation and EZH2 gain is rather prevalent in melanoma. Our findings provided evidence for the feasibility of combination therapy with EZH2 and BRAF inhibitors in melanoma with concurrent BRAF V600E mutation and EZH2 gain.

A tailored approach to BRAF and MLH1 methylation testing in a universal screening program for Lynch syndrome.

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Adar, Tomer; Rodgers, Linda H; Shannon, Kristen M; Yoshida, Makoto; Ma, Tianle; Mattia, Anthony; Lauwers, Gregory Y; Iafrate, Anthony J; Chung, Daniel C

2017-03-01

To determine the correlation between BRAF genotype and MLH1 promoter methylation in a screening program for Lynch syndrome (LS), a universal screening program for LS was established in two medical centers. Tumors with abnormal MLH1 staining were evaluated for both BRAF V600E genotype and MLH1 promoter methylation. Tumors positive for both were considered sporadic, and genetic testing was recommended for all others. A total 1011 colorectal cancer cases were screened for Lynch syndrome, and 148 (14.6%) exhibited absent MLH1 immunostaining. Both BRAF and MLH1 methylation testing were completed in 126 cases. Concordant results (both positive or both negative) were obtained in 86 (68.3%) and 16 (12.7%) cases, respectively, with 81% concordance overall. The positive and negative predictive values for a BRAF mutation in predicting MLH1 promoter methylation were 98.9% and 41%, respectively, and the negative predictive value fell to 15% in patients ≥70 years old. Using BRAF genotyping as a sole test to evaluate cases with absent MLH1 staining would have increased referral rates for genetic testing by 2.3-fold compared with MLH1 methylation testing alone (31% vs 13.5%, respectively, PMLH1 methylation testing for BRAF wild-type cases only would significantly decrease the number of methylation assays performed and reduce the referral rate for genetic testing to 12.7%. A BRAF mutation has an excellent positive predictive value but poor negative predictive value in predicting MLH1 promoter methylation. A hybrid use of these tests may reduce the number of low-risk patients referred to genetic counseling and facilitate wider implementation of Lynch syndrome screening programs.

Immunohistochemical detection of the BRAF V600E mutation in papillary thyroid carcinoma. Evaluation against real-time polymerase chain reaction.

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Paja Fano, Miguel; Ugalde Olano, Aitziber; Fuertes Thomas, Elena; Oleaga Alday, Amelia

2017-02-01

The BRAF V600E mutation is the most common genetic change in papillary thyroid carcinoma and is associated with a poorer clinical course. Usual methods for its study (DNA sequencing or molecular test based on PCR) are expensive and time-consuming. Recently, immunohistochemistry (IHC) for BRAF mutation has been introduced. To compare the results of IHC and real time PCR (RT-PCR) in the detection of BRAF V600E mutation in papillary thyroid carcinoma. Analysis of clinical and pathological differences depending on RT-PCR results is included. A prospective study was performed in 82 consecutive samples, 54 of them taken through a core needle biopsy. IHC was performed on tissue fixed for 24hours with 10% neutral formalin using the anti-BRAF V600E (VE-1) mouse monoclonal primary antibody and was rated as positive or negative. DNA was extracted from formalin-fixed, paraffin-embedded tissues by manual microdissection, and BRAF mutation was detected by RT-PCR using the Cobas® 4800 BRAF V600 mutation test (Roche). Both techniques were concordant in 81 cases, and BRAF was positive in 49. Discordance appeared in a follicular variant showing positive IHC and negative RT-PCR, attributed to histological heterogeneity. Cost of materials for IHC was less than half of the cost for RT-PCR. IHC appears to be a reliable, economical and easily available alternative to molecular biology techniques for routine detection of the BRAF V600E mutation in papillary thyroid carcinoma patients, provided optimal fixation conditions are used. It may be a useful technique in hospitals with no access to molecular biology techniques. Copyright © 2017 SEEN. Publicado por Elsevier España, S.L.U. All rights reserved.

Low incidence of minor BRAF V600 mutation-positive subclones in primary and metastatic melanoma determined by sensitive and quantitative real-time PCR

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Kielsgaard Kristensen, Thomas; Clemmensen, Ole; Hoejberg, Lise

2013-01-01

BRAF V600 mutation is an important biological marker for therapeutic guidance in melanoma, where mutation-positive cases are candidates for therapy targeting mutant B-Raf. Recent studies showing intratumor variation in BRAF mutation status have caused concern that sensitive mutation analysis can ...

The value of the repeated examination of BRAF V600E mutation status in diagnostics of papillary thyroid cancer.

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Beiša, Augustas; Beiša, Virgilijus; Stoškus, Mindaugas; Ostanevičiūtė, Elvyra; Griškevičius, Laimonas; Strupas, Kęstutis

2016-01-01

Nodular thyroid disease is one of the most frequently diagnosed pathologies of the adult population in iodine-deficient regions. Approximately 30% of thyroid aspirates are classified as nondiagnostic/unsatisfactory or indeterminate. However, patients with indeterminate cytology still undergo surgery. The object of this study was to determine the diagnostic value of re-examining the BRAF V600E mutation in papillary thyroid carcinoma patients. All patients underwent ultrasound guided fine-needle aspiration of a thyroid nodule. They were assigned to one of the four groups (indeterminate or positive for malignant cells) of the Bethesda System for Reporting Thyroid Cytopathology. Genetic investigation of the BRAF V600E mutation was performed for all of the fine-needle aspiration cytology specimens. All of the patients underwent surgery. Subsequently, histological investigation of the removed tissues was performed. Additional analysis of the BRAF V600E mutation from the histology specimen was then performed for the initially BRAF-negative cases. Two hundred and fourteen patients were involved in the study. One hundred and six (49.53%) patients were diagnosed with thyroid cancer. Of these 106 patients, 95 (89.62%) patients were diagnosed with papillary thyroid cancer. The BRAF V600E mutation was positive in 62 (65.26%) and negative in 33 (34.74%) histologically confirmed papillary thyroid cancer cases. After the genetic investigation, a total of 74 (77.89%) papillary thyroid cancer cases were positive for the BRAF V600E mutation and 21 (22.11%) were negative. Repeated examination of the BRAF V600E mutation status in the fine-needle aspiration may potentially increase the sensitivity of papillary thyroid cancer diagnostics.

The CIMP Phenotype in BRAF Mutant Serrated Polyps from a Prospective Colonoscopy Patient Cohort

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Winnie C. Fernando

2014-01-01

Full Text Available Colorectal cancers arising via the serrated pathway are often associated with BRAF V600E mutation, CpG island methylator phenotype (CIMP, and microsatellite instability. Previous studies have shown a strong association between BRAF V600E mutation and serrated polyps. This study aims to evaluate CIMP status of all the serrated polyp subtypes and its association with functionally important genes such as MLH1, p16, and IGFBP7. CIMP status and methylation were evaluated using the real-time based MethyLight assay in 154 serrated polyps and 63 conventional adenomas. Results showed that CIMP-high serrated polyps were strongly associated with BRAF mutation and proximal colon. CIMP-high was uncommon in conventional adenomas (1.59%, occurred in 8.25% of hyperplastic polyps (HPs, and became common in sessile serrated adenomas (SSAs (51.43%. MLH1 methylation was mainly observed in the proximal colon and was significantly associated with BRAF mutation and CIMP-high. The number of samples methylated for p16 and IGFBP7 was the highest in SSAs. The methylation panel we used to detect CIMP is highly specific for CIMP-high cancers. With this panel, we demonstrate that CIMP-high is much more common in SSAs than HPs. This suggests that CIMP-high correlates with increased risk of malignant transformation which was also observed in methylation of functionally important genes.

The CIMP Phenotype in BRAF Mutant Serrated Polyps from a Prospective Colonoscopy Patient Cohort.

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Fernando, Winnie C; Miranda, Mariska S; Worthley, Daniel L; Togashi, Kazutomo; Watters, Dianne J; Leggett, Barbara A; Spring, Kevin J

2014-01-01

Colorectal cancers arising via the serrated pathway are often associated with BRAF V600E mutation, CpG island methylator phenotype (CIMP), and microsatellite instability. Previous studies have shown a strong association between BRAF V600E mutation and serrated polyps. This study aims to evaluate CIMP status of all the serrated polyp subtypes and its association with functionally important genes such as MLH1, p16, and IGFBP7. CIMP status and methylation were evaluated using the real-time based MethyLight assay in 154 serrated polyps and 63 conventional adenomas. Results showed that CIMP-high serrated polyps were strongly associated with BRAF mutation and proximal colon. CIMP-high was uncommon in conventional adenomas (1.59%), occurred in 8.25% of hyperplastic polyps (HPs), and became common in sessile serrated adenomas (SSAs) (51.43%). MLH1 methylation was mainly observed in the proximal colon and was significantly associated with BRAF mutation and CIMP-high. The number of samples methylated for p16 and IGFBP7 was the highest in SSAs. The methylation panel we used to detect CIMP is highly specific for CIMP-high cancers. With this panel, we demonstrate that CIMP-high is much more common in SSAs than HPs. This suggests that CIMP-high correlates with increased risk of malignant transformation which was also observed in methylation of functionally important genes.

BRAF and RAS oncogenes regulate Rho GTPase pathways to mediate migration and invasion properties in human colon cancer cells: a comparative study

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Shirasawa Senji

2011-09-01

Full Text Available Abstract Background Colorectal cancer is a common disease that involves genetic alterations, such as inactivation of tumour suppressor genes and activation of oncogenes. Among them are RAS and BRAF mutations, which rarely coexist in the same tumour. Individual members of the Rho (Ras homology GTPases contribute with distinct roles in tumour cell morphology, invasion and metastasis. The aim of this study is to dissect cell migration and invasion pathways that are utilised by BRAFV600E as compared to KRASG12V and HRASG12V oncoproteins. In particular, the role of RhoA (Ras homolog gene family, member A, Rac1 (Ras-related C3 botulinum toxin substrate 1 and Cdc42 (cell division cycle 42 in cancer progression induced by each of the three oncogenes is described. Methods Colon adenocarcinoma cells with endogenous as well as ectopically expressed or silenced oncogenic mutations of BRAFV600E, KRASG12V and HRASG12V were employed. Signalling pathways and Rho GTPases were inhibited with specific kinase inhibitors and siRNAs. Cell motility and invasion properties were correlated with cytoskeletal properties and Rho GTPase activities. Results Evidence presented here indicate that BRAFV600E significantly induces cell migration and invasion properties in vitro in colon cancer cells, at least in part through activation of RhoA GTPase. The relationship established between BRAFV600E and RhoA activation is mediated by the MEK-ERK pathway. In parallel, KRASG12V enhances the ability of colon adenocarcinoma cells Caco-2 to migrate and invade through filopodia formation and PI3K-dependent Cdc42 activation. Ultimately increased cell migration and invasion, mediated by Rac1, along with the mesenchymal morphology obtained through the Epithelial-Mesenchymal Transition (EMT were the main characteristics rendered by HRASG12V in Caco-2 cells. Moreover, BRAF and KRAS oncogenes are shown to cooperate with the TGFβ-1 pathway to provide cells with additional transforming

Dabrafenib and Trametinib Treatment in Patients With Locally Advanced or Metastatic BRAF V600-Mutant Anaplastic Thyroid Cancer

NARCIS (Netherlands)

Subbiah, Vivek; Kreitman, Robert J; Wainberg, Zev A; Cho, Jae Yong; Schellens, Jan H M; Soria, Jean Charles; Wen, Patrick Y; Zielinski, Christoph; Cabanillas, Maria E; Urbanowitz, Gladys; Mookerjee, Bijoyesh; Wang, Dazhe; Rangwala, Fatima; Keam, Bhumsuk

2018-01-01

Purpose We report the efficacy and safety of dabrafenib (BRAF inhibitor) and trametinib (MEK inhibitor) combination therapy in BRAF V600E-mutated anaplastic thyroid cancer, a rare, aggressive, and highly lethal malignancy with poor patient outcomes and no systemic therapies with clinical benefit.

BRAF inhibition improves tumor recognition by the immune system

DEFF Research Database (Denmark)

Donia, Marco; Fagone, Paolo; Nicoletti, Ferdinando

2012-01-01

to be poorly efficient. By characterizing the immunological interactions between T cells and cancer cells in clinical material as well as the influence of the FDA-approved BRAF inhibitor vemurafenib on the immune system, we aimed at unraveling new strategies to expand the efficacy of adoptive T-cell transfer...

Concordance in BRAF V600E status over time in malignant melanoma and corresponding metastases

DEFF Research Database (Denmark)

Nielsen, Line Bjerrehave; Dabrosin, Nina; Sloth, Karen

2017-01-01

of inter-tumour heterogeneity between the primary tumour and the corresponding metastases, time as a factor was also investigated. METHODS: In total, 227 samples from 224 melanoma patients were analysed using both the Cobas® 4800 BRAF V600 Mutation test and IHC anti-BRAF V600E staining. In 82 primary......AIMS: The present study analysed the usability of an immunohistochemical (IHC) analysis compared to a frequently used mutation detection analysis and examined the extent of intra- and inter-tumour heterogeneity of BRAF V600E in primary tumours and their corresponding metastases. In the development...... tumours and 224 corresponding metastases, the extent of inter- and intra-tumour heterogeneity was investigated using IHC staining. RESULTS: In 15 cases, disagreement between IHC analysis and the Cobas test was seen. In all but one of the examined patients, homogeneity between the primary tumour...

Extended Antitumor Responseof a BRAF V600E Papillary Thyroid Carcinoma to Vemurafenib

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Siraj M. Ali

2014-05-01

Full Text Available Context: For patients with metastatic papillary thyroid carcinoma (PTC refractory to radioactive iodine (RAI treatment, systemic chemotherapy has limited efficacy. Such tumors frequently harbor BRAF V600E, and this alteration may predict responsiveness to vemura-fenib treatment. Objective: We report a metastatic PTC patient refractory to RAI treatment that underwent genomic profiling by next-generation sequencing. The sole genomic alteration identified was BRAF V600E on a near diploid genome with trisomy 1q. With vemurafenib treatment, the patient experienced a dramatic radiographic and clinical improvement, with the duration of an ongoing antitumor response exceeding 23 months. Design: Hybridization capture of 3,769 exons of 236 cancer-related genes and the introns of 19 genes frequently rearranged in cancer was applied to >50 ng of DNA extracted from a formalin-fixed, paraffin-embedded biopsy of a lymph node containing metastatic PTC and was sequenced to a high, uniform coverage of ×616. Results: A BRAF V600E alteration was identified with no other somatic genomic alterations present within a near diploid tumor genome. The patient initially received vemurafenib at 960 mg twice daily that was reduced to 480 mg twice daily due to rash and diarrhea and has experienced an ongoing antitumor response exceeding 23 months by both PET-CT and dedicated CT imaging. Conclusions: Genomic profiling in metastatic, RAI-refractory PTC can reveal a targetable BRAF V600E alteration without compounding somatic alterations, and such patients may derive a more prolonged benefit from vemurafenib treatment. Prospective clinical trials are ongoing to confirm our preliminary observation.

Patient and tumor characteristics and BRAF and KRAS mutations in colon cancer, NCCTG/Alliance N0147.

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Gonsalves, Wilson I; Mahoney, Michelle R; Sargent, Daniel J; Nelson, Garth D; Alberts, Steven R; Sinicrope, Frank A; Goldberg, Richard M; Limburg, Paul J; Thibodeau, Stephen N; Grothey, Axel; Hubbard, Joleen M; Chan, Emily; Nair, Suresh; Berenberg, Jeffrey L; McWilliams, Robert R

2014-07-01

KRAS and BRAF (V600E) mutations are important predictive and prognostic markers, respectively, in colon cancer, but little is known about patient and clinical factors associated with them. Two thousand three hundred twenty-six of 3397 patients in the N0147 phase III adjuvant trial for stage III colon cancer completed a patient questionnaire. Primary tumors were assessed for KRAS and BRAF (V600E) mutations and defective mismatch repair (dMMR) status. Logistic regression models and categorical data analysis were used to identify associations of patient and tumor characteristics with mutation status. All statistical tests were two-sided. KRAS (35%) and BRAF (V600E) (14%) mutations were nearly mutually exclusive. KRAS mutations were more likely to be present in patients without a family history of colon cancer and never smokers. Tumors with KRAS mutations were less likely to have dMMR (odds ratio [OR] = 0.21; 95% confidence interval [CI] = 0.15 to 0.31; P characteristics are associated with KRAS and BRAF (V600E) mutations. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Improvement of the quality of BRAF testing in melanomas with nationwide external quality assessment, for the BRAF EQA group

International Nuclear Information System (INIS)

Emile, Jean-François; Hofman, Paul; Sabourin, Jean-Christophe; Laurent-Puig, Pierre; Tisserand, Julie; Bergougnoux, Loic; Nowak, Frédérique; Faucher, Gladwys; Surel, Sylvie; Lamy, Aude; Lecorre, Delphine; Helias-Rodzewicz, Zofia

2013-01-01

Knowledge about tumour gene mutation status is essential for the treatment of increasing numbers of cancer patients, and testing quality has a major impact on treatment response and cost. In 2012, 4,629 tests for BRAF p.V600 were performed in France, in patients with melanomas. Two batches of unstained melanoma sections were sent, in May and November 2012, to the 46 laboratories supported by the French National Institute of Cancer (INCa). An external quality assessment (EQA) evaluated mutation status, response times and compliance with INCa recommendations. All the French laboratories involved in testing participated in the EQA. Fourteen different methods were used to detect BRAF mutations, most consisting of combinations of in-house techniques. False responses were noted in 25/520 cases (4.8%), 11 of which concerned confusion between p.V600E and p.V600K. Thus, 2.7% of responses would have led to inappropriate treatment. Within six months, mean response times decreased from 22 to 12 days (P<0.001), and the percentage of samples evaluated by a pathologist for tumour cell content increased, from 75.2% to 96.9% (P<0.001). Despite the use of non-certified methods, the false response rate was low. Nationwide EQA can improve the quality of molecular pathology tests on tumours

The Prognostic Influence of BRAF Mutation and other Molecular, Clinical and Laboratory Parameters in Stage IV Colorectal Cancer.

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Karadima, Maria L; Saetta, Angelica A; Chatziandreou, Ilenia; Lazaris, Andreas C; Patsouris, Efstratios; Tsavaris, Nikolaos

2016-10-01

Our aim was to evaluate the predictive and prognostic influence of BRAF mutation and other molecular, clinical and laboratory parameters in stage IV colorectal cancer (CRC). 60 patients were included in this retrospective analysis, and 17 variables were examined for their relation with treatment response and survival. KRAS mutation was identified in 40.3 % of cases, BRAF and PIK3CA in 8.8 % and 10.5 % respectively. 29.8 % of patients responded to treatment. Median survival time was 14.3 months. Weight loss, fever, abdominal metastases, blood transfusion, hypoalbuminaimia, BRAF and PIK3CA mutations, CRP and DNA Index were associated with survival. In multivariate analysis, male patients had 3.8 times higher probability of response, increased DNA Index was inversely correlated with response and one unit raise of DNA Index augmented 6 times the probability of death. Our findings potentiate the prognostic role of BRAF, PIK3CA mutations and ploidy in advanced CRC.

BRAF mutation and anaplasia may be predictive factors of progression-free survival in adult pleomorphic xanthoastrocytoma.

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Tabouret, E; Bequet, C; Denicolaï, E; Barrié, M; Nanni, I; Metellus, P; Dufour, Henri; Chinot, O; Figarella-Branger, D

2015-12-01

Pleomorphic xanthoastrocytoma (PXA) is a rare, low-grade glioma that frequently occurs in pediatric patients. To analyze adult patients diagnosed with PXA and to search for pathological and molecular markers of diagnosis and prognosis. We retrospectively included patients older than 16 years with PXA who were referred to our institution between October 2003 and September 2013. All pathological diagnoses were reviewed by a neuropathologist. Histological characteristics and immunostaining of GFAP, OLIG2, neurofilament, CD34, Ki67, p53, p16, and IDH1 R132H were analyzed. The following molecular alterations were analyzed: mutations of IDH1/2, BRAF and the histone H3.3 and the EGFR amplification. Clinical data, treatment modalities, and patient outcome were recorded. We identified 16 adult patients with reviewed PXA diagnosis. No IDH neither histone H3.3 mutations were found; BRAF V600E mutation was recorded in six patients. Ten patients presented with anaplastic features. BRAF mutations were associated with lower Ki67, OLIG2 expression, and lack of p16 expression. Median PFS and OS were 41.5 months (95% CI: 11.4-71.6) and 71.4 months (95% CI: 15.5-127.3), respectively. BRAF mutation tended to be associated with greater PFS (p = 0.051), whereas anaplastic features were associated with minimal PFS (p = 0.042). PXA in adults PXA may present features distinct from pediatric PXA. Anaplastic features and BRAF mutation may potentially identify specific subgroups with distinct prognoses. Copyright © 2015 Elsevier Ltd. All rights reserved.

BRAF inhibition is associated with increased clonality in tumor-infiltrating lymphocytes

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Cooper, Zachary A; Frederick, Dennie T; Juneja, Vikram R; Sullivan, Ryan J; Lawrence, Donald P; Piris, Adriano; Sharpe, Arlene H; Fisher, David E; Flaherty, Keith T; Wargo, Jennifer A

2013-01-01

There have been significant advances with regard to BRAF-targeted therapies against metastatic melanoma. However, the majority of patients receiving BRAF inhibitors (BRAFi) manifest disease progression within a year. We have recently shown that melanoma patients treated with BRAFi exhibit an increase in melanoma-associated antigens and in CD8+ tumor-infiltrating lymphocytes in response to therapy. To characterize such a T-cell infiltrate, we analyzed the complementarity-determining region 3 (CDR3) of rearranged T-cell receptor (TCR) β chain-coding genes in tumor biopsies obtained before the initiation of BRAFi and 10–14 d later. We observed an increase in the clonality of tumor-infiltrating lymphocytes in 7 of 8 patients receiving BRAFi, with a statistically significant 21% aggregate increase in clonality. Over 80% of individual T-cell clones detected after initiation of BRAFi treatment were new clones. Interestingly, the comparison of tumor infiltrates with clinical responses revealed that patients who had a high proportion of pre-existing dominant clones after the administration of BRAFi responded better to therapy than patients who had a low proportion of such pre-existing dominant clones following BRAFi. These data suggest that although the inhibition of BRAF in melanoma patients results in tumor infiltration by new lymphocytes, the response to treatment appears to be related to the presence of a pre-existing population of tumor-infiltrating T-cell clones. PMID:24251082

Cost-effectiveness of treatment strategies for BRAF-mutated metastatic melanoma.

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Patti Curl

Full Text Available Genetically-targeted therapies are both promising and costly advances in the field of oncology. Several treatments for metastatic melanoma with a mutation in the BRAF gene have been approved. They extend life but are more expensive than the previous standard of care (dacarbazine. Vemurafenib, the first drug in this class, costs $13,000 per month ($207,000 for a patient with median survival. Patients failing vemurafenib are often given ipilimumab, an immunomodulator, at $150,000 per course. Assessment of cost-effectiveness is a valuable tool to help navigate the transition toward targeted cancer therapy.We performed a cost-utility analysis to compare three strategies for patients with BRAF+ metastatic melanoma using a deterministic expected-value decision tree model to calculate the present value of lifetime costs and quality-adjusted life years (QALYs for each strategy. We performed sensitivity analyses on all variables.In the base case, the incremental cost-effectiveness ratio (ICER for vemurafenib compared with dacarbazine was $353,993 per QALY gained (0.42 QALYs added, $156,831 added. The ICER for vemurafenib followed by ipilimumab compared with vemurafenib alone was $158,139. In sensitivity analysis, treatment cost had the largest influence on results: the ICER for vemurafenib versus dacarbazine dropped to $100,000 per QALY gained with a treatment cost of $3600 per month.The cost per QALY gained for treatment of BRAF+ metastatic melanoma with vemurafenib alone or in combination exceeds widely-cited thresholds for cost-effectiveness. These strategies may become cost-effective with lower drug prices or confirmation of a durable response without continued treatment.

A Combined Pharmacophore Modeling, 3D QSAR and Virtual Screening Studies on Imidazopyridines as B-Raf Inhibitors

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Huiding Xie

2015-05-01

Full Text Available B-Raf kinase is an important target in treatment of cancers. In order to design and find potent B-Raf inhibitors (BRIs, 3D pharmacophore models were created using the Genetic Algorithm with Linear Assignment of Hypermolecular Alignment of Database (GALAHAD. The best pharmacophore model obtained which was used in effective alignment of the data set contains two acceptor atoms, three donor atoms and three hydrophobes. In succession, comparative molecular field analysis (CoMFA and comparative molecular similarity indices analysis (CoMSIA were performed on 39 imidazopyridine BRIs to build three dimensional quantitative structure-activity relationship (3D QSAR models based on both pharmacophore and docking alignments. The CoMSIA model based on the pharmacophore alignment shows the best result (q2 = 0.621, r2pred = 0.885. This 3D QSAR approach provides significant insights that are useful for designing potent BRIs. In addition, the obtained best pharmacophore model was used for virtual screening against the NCI2000 database. The hit compounds were further filtered with molecular docking, and their biological activities were predicted using the CoMSIA model, and three potential BRIs with new skeletons were obtained.

A Combined Pharmacophore Modeling, 3D QSAR and Virtual Screening Studies on Imidazopyridines as B-Raf Inhibitors.

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Xie, Huiding; Chen, Lijun; Zhang, Jianqiang; Xie, Xiaoguang; Qiu, Kaixiong; Fu, Jijun

2015-05-29

B-Raf kinase is an important target in treatment of cancers. In order to design and find potent B-Raf inhibitors (BRIs), 3D pharmacophore models were created using the Genetic Algorithm with Linear Assignment of Hypermolecular Alignment of Database (GALAHAD). The best pharmacophore model obtained which was used in effective alignment of the data set contains two acceptor atoms, three donor atoms and three hydrophobes. In succession, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were performed on 39 imidazopyridine BRIs to build three dimensional quantitative structure-activity relationship (3D QSAR) models based on both pharmacophore and docking alignments. The CoMSIA model based on the pharmacophore alignment shows the best result (q(2) = 0.621, r(2)(pred) = 0.885). This 3D QSAR approach provides significant insights that are useful for designing potent BRIs. In addition, the obtained best pharmacophore model was used for virtual screening against the NCI2000 database. The hit compounds were further filtered with molecular docking, and their biological activities were predicted using the CoMSIA model, and three potential BRIs with new skeletons were obtained.

Braf, Kras and Helicobacter pylori epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer.

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Sabry, Dina; Ahmed, Rasha; Abdalla, Sayed; Fathy, Wael; Eldemery, Ahmed; Elamir, Azza

2016-06-01

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.

BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

NARCIS (Netherlands)

E.H.J. van Roon (Eddy); A. Boot (Arnoud); A.A. Dihal (Ashwin); R.F. Ernst (Robert); T. van Wezel (Tom); H. Morreau (Hans); J.M. Boer (Judith)

2013-01-01

textabstractBackground: Cancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have

Dabrafenib Treatment in a Patient with an Epithelioid Glioblastoma and BRAF V600E Mutation

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Garry Ceccon

2018-04-01

Full Text Available Novel therapeutic targets in malignant glioma patients are urgently needed. Point mutations of the v-Raf murine sarcoma viral oncogene homolog B (BRAF gene occur predominantly in melanoma patients, but may also occur in gliomas. Thus, this is a target of great interest for this group of patients. In a nine-year-old male patient, an anaplastic astrocytoma in the left temporoparietal region was diagnosed histologically. After first- and second-line treatment, a malignant progression to a secondary glioblastoma was observed ten years after the initial diagnosis. Within the following seven years, all other conventional treatment options were exhausted. At this time point, recurrent tumor histology revealed an epithelioid glioblastoma, without a mutation in the isocitrate dehydrogenase gene (IDH wild-type. In order to identify a potential target for an experimental salvage therapy, mutational tumor analysis showed a BRAF V600E mutation. Consecutively, dabrafenib treatment was initiated. The patient remained clinically stable, and follow-up magnetic resonance images (MRI were consistent with “Stable Disease” according to the Response Assessment in Neuro-Oncology Working Group (RANO criteria for the following ten months until tumor progression was detected. The patient died 16 months after dabrafenib treatment initiation. Particularly in younger glioma patients as well as in patients with an epithelioid glioblastoma, screening for a V600E BRAF mutation is promising since, in these cases, targeted therapy with BRAF inhibitors seems to be a useful salvage treatment option.

Mutational analysis of cutaneous squamous cell carcinomas and verrucal keratosis in patients taking BRAF inhibitors.

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Anforth, Rachael; Tembe, Varsha; Blumetti, Tatiana; Fernandez-Peñas, Pablo

2012-09-01

B-RAF inhibitors (BRAFi) have been shown to improve rates of overall and progression-free survival in patients with stage IV metastatic melanoma positive for the BRAF V600E mutation. However, the main drawback is the development of verrucal keratosis (hyperkeratotic papules with verruca-like characteristics with benign histological findings) and cutaneous squamous cell carcinomas (cuSCC). We have found upstream mutations in RAS as well as PIK3CA in both verrucal keratosis and cuSCC. This suggests that verrucal keratosis is an early clinical presentation of cuSCC in patients on BRAFi. © 2012 John Wiley & Sons A/S.

The prognostic value of BRAF mutation and lymph node metastases in patients with papillary thyroid cancer

International Nuclear Information System (INIS)

Takacsova, E.

2015-01-01

Full text of publication follows. Aim: To assess the prognostic significance of BRAF mutation and lymph node metastases in patients with papillary thyroid cancer. Method: between January 2010 and March 2012, we retrospectively analysed 172 patients after total thyroidectomy with neck dissection for papillary thyroid cancer. Mutation in the BRAF gene (V600E) was assessed in all of the enrolled patients. According to TNM classification, 56 (33%) patients were in low risk group and 116 (67%) patients in high risk group. Among high risk group, 105 out of 116 (90%) patients were presented with lymph node metastases. Thyroid ablation with radioactive iodine was performed in all of the patients enrolled in the study. Persistent or recurrent disease was diagnosed in 42 (24%) patients during radioiodine therapy. Results and conclusion: BRAF mutation did not appear to be significant unfavourable prognostic factor in our cohort: it was present in 55% of low risk patients, in 48% of high risk patients and in 46% of patients with lymph node metastases. In patients with persistent or recurrent disease, BRAF mutation was found in 48%. But we confirmed that the presence of lymph node metastases in time of initial surgery (61% of all patients) appeared to be significant: neck dissection enabled a more precise classification of patients into the high risk group - up to 95% of patients with persistent or recurrent disease diagnosed during radioiodine therapy belonged to the high risk group in the beginning of therapy. (author)

Evaluation and identification of factors related to KRAS and BRAF gene mutations in colorectal cancer: A meta-analysis

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Li Lin

2016-01-01

Conclusion: The meta-analysis reveals that KRAS has a slightly higher mutation rate in MSI-L/MSS tumors. Moreover, BRAF mutations have higher detection rates in right-sided colorectal cancer, which suggests that BRAF mutations are likely in CIMP-H tumors. Therefore, based on these findings, the molecular diagnostic tests to be conducted in colorectal cancer patients can be determined according to the location/clinical features of the tumor.

Survival According to BRAF-V600 Tumor Mutations – An Analysis of 437 Patients with Primary Melanoma

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Meckbach, Diana; Bauer, Jürgen; Pflugfelder, Annette; Meier, Friedegund; Busch, Christian; Eigentler, Thomas K.; Capper, David; von Deimling, Andreas; Mittelbronn, Michel; Perner, Sven; Ikenberg, Kristian; Hantschke, Markus; Büttner, Petra; Garbe, Claus; Weide, Benjamin

2014-01-01

The prognostic impact of BRAF-V600 tumor mutations in stage I/II melanoma patients has not yet been analyzed in detail. We investigated primary tumors of 437 patients diagnosed between 1989 and 2006 by Sanger sequencing. Mutations were detected in 38.7% of patients and were associated with age, histological subtype as well as mitotic rate. The mutational rate was 36.7% in patients with disease-free course and 51.7% in those with subsequent distant metastasis (p = 0.031). No difference in overall survival (p = 0.119) but a trend for worse distant-metastasis-free survival (p = 0.061) was observed in BRAF mutant compared to BRAF wild-type patients. Independent prognostic factors for overall survival were tumor thickness, mitotic rate and ulceration. An interesting significant prognostic impact was observed in patients with tumor thickness of 1 mm or less, with the mutation present in 6 of 7 patients dying from melanoma. In conclusion, no significant survival differences were found according to BRAF-V600 tumor mutations in patients with primary melanoma but an increasing impact of the mutational status was observed in the subgroup of patients with tumor thickness of 1 mm or less. A potential role of the mutational status as a prognostic factor especially in this subgroup needs to be investigated in larger studies. PMID:24475086

Preappointment testing for BRAF/KIT mutation in advanced melanoma: a model in molecular data delivery for individualized medicine.

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Mounajjed, Taofic; Brown, Char L; Stern, Therese K; Bjorheim, Annette M; Bridgeman, Andrew J; Rumilla, Kandelaria M; McWilliams, Robert R; Flotte, Thomas J

2014-11-01

The emergence of individualized medicine is driven by developments in precision diagnostics, epitomized by molecular testing. Because treatment decisions are being made based on such molecular data, data management is gaining major importance. Among data management challenges, creating workflow solutions for timely delivery of molecular data has become pivotal. This study aims to design and implement a scalable process that permits preappointment BRAF/KIT mutation analysis in melanoma patients, allowing molecular results necessary for treatment plans to be available before the patient's appointment. Process implementation aims to provide a model for efficient molecular data delivery for individualized medicine. We examined the existing process of BRAF/KIT testing in melanoma patients visiting our institution for oncology consultation. We created 5 working groups, each designing a specific segment of an alternative process that would allow preappointment BRAF/KIT testing and delivery of results. Data were captured and analyzed to evaluate the success of the alternative process. For 1 year, 35 (59%) of 55 patients had prior BRAF/KIT testing. The remaining 20 patients went through the new process of preappointment testing; results were available at the time of appointment for 12 patients (overall preappointment results availability, 85.5%). The overall process averaged 13.4 ± 4.7 days. In conclusion, we describe the successful implementation of a scalable workflow solution that permits preappointment BRAF/KIT mutation analysis and result delivery in melanoma patients. This sets the stage for further applications of this model to other conditions, answering an increasing demand for robust delivery of molecular data for individualized medicine. Copyright © 2014 Elsevier Inc. All rights reserved.

Malignant melanoma arising from a perianal fistula and harbouring a BRAF gene mutation: a case report

International Nuclear Information System (INIS)

Martinez-Cadenas, Conrado; Lozoya, Rafael; Boldó, Enrique; Bosch, Nuria; Peñas, Lucas; Flores-Couce, Esther; Ochoa, Enrique; Munárriz, Javier; Aracil, Juan P; Tajahuerce, Marcos; Royo, Ramón

2011-01-01

Melanoma of the anal region is a very uncommon disease, accounting for only 0.2-0.3% of all melanoma cases. Mutations of the BRAF gene are usually absent in melanomas occurring in this region as well as in other sun-protected regions. The development of a tumour in a longstanding perianal fistula is also extremely rare. More frequent is the case of a tumour presenting as a fistula, that is, the fistula being a consequence of the cancerous process, although we have found only two cases of fistula-generating melanomas reported in the literature. Here we report the case of a 38-year-old male who presented with a perianal fistula of four years of evolution. Histopathological examination of the fistulous tract confirmed the presence of malignant melanoma. Due to the small size and the central location of the melanoma inside the fistulous tract, we believe the melanoma reported here developed in the epithelium of the fistula once the latter was already formed. Resected sentinel lymph nodes were negative and the patient, after going through a wide local excision, remains disease-free nine years after diagnosis. DNA obtained from melanoma tissue was analysed by automated direct sequencing and the V600E (T1799A) mutation was detected in exon 15 of the BRAF gene. Since fistulae experience persistent inflammation, the fact that this melanoma harbours a BRAF mutation strengthens the view that oxidative stress caused by inflammatory processes plays an important role in the genesis of BRAF gene mutations

Malignant melanoma arising from a perianal fistula and harbouring a BRAF gene mutation: a case report

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Tajahuerce Marcos

2011-08-01

Full Text Available Abstract Background Melanoma of the anal region is a very uncommon disease, accounting for only 0.2-0.3% of all melanoma cases. Mutations of the BRAF gene are usually absent in melanomas occurring in this region as well as in other sun-protected regions. The development of a tumour in a longstanding perianal fistula is also extremely rare. More frequent is the case of a tumour presenting as a fistula, that is, the fistula being a consequence of the cancerous process, although we have found only two cases of fistula-generating melanomas reported in the literature. Case Presentation Here we report the case of a 38-year-old male who presented with a perianal fistula of four years of evolution. Histopathological examination of the fistulous tract confirmed the presence of malignant melanoma. Due to the small size and the central location of the melanoma inside the fistulous tract, we believe the melanoma reported here developed in the epithelium of the fistula once the latter was already formed. Resected sentinel lymph nodes were negative and the patient, after going through a wide local excision, remains disease-free nine years after diagnosis. DNA obtained from melanoma tissue was analysed by automated direct sequencing and the V600E (T1799A mutation was detected in exon 15 of the BRAF gene. Conclusion Since fistulae experience persistent inflammation, the fact that this melanoma harbours a BRAF mutation strengthens the view that oxidative stress caused by inflammatory processes plays an important role in the genesis of BRAF gene mutations.

PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling

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Shrestha, Yashaswi; Schafer, Eric J.; Boehm, Jesse S.; Thomas, Sapana R.; He, Frank; Du, Jinyan; Wang, Shumei; Barretina, Jordi; Weir, Barbara A.; Zhao, Jean J.; Polyak, Kornelia; Golub, Todd R.; Beroukhim, Rameen; Hahn, William C.

2011-01-01

Activating mutations in the RAS family or BRAF frequently occur in many types of human cancers but are rarely detected in breast tumors. However, activation of the RAS-RAF-MEK-ERK Mitogen-Activated Protein Kinase (MAPK) pathway is commonly observed in human breast cancers, suggesting that other genetic alterations lead to activation of this signaling pathway. To identify breast cancer oncogenes that activate the MAPK pathway, we screened a library of human kinases for their ability to induce ...

FREQUENT SUBCLINICAL MACULAR CHANGES IN COMBINED BRAF/MEK INHIBITION WITH HIGH-DOSE HYDROXYCHLOROQUINE AS TREATMENT FOR ADVANCED METASTATIC BRAF MUTANT MELANOMA: Preliminary Results From a Phase I/II Clinical Treatment Trial.

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Nti, Akosua A; Serrano, Leona W; Sandhu, Harpal S; Uyhazi, Katherine E; Edelstein, Ilaina D; Zhou, Elaine J; Bowman, Scott; Song, Delu; Gangadhar, Tara C; Schuchter, Lynn M; Mitnick, Sheryl; Huang, Alexander; Nichols, Charles W; Amaravadi, Ravi K; Kim, Benjamin J; Aleman, Tomas S

2018-01-10

To assess the potential ocular toxicity of a combined BRAF inhibition (BRAFi) + MEK inhibition (MEKi) + hydroxychloroquine (HCQ) regime used to treat metastatic BRAF mutant melanoma. Patients with stage IV metastatic melanoma and BRAF V600E mutations (n = 11, 31-68 years of age) were included. Treatment was with oral dabrafenib, 150 mg bid, trametinib, 2 mg/day, and HCQ, 400 mg to 600 mg bid. An ophthalmic examination, spectral domain optical coherence tomography, near-infrared and short-wavelength fundus autofluorescence, and static perimetry were performed at baseline, 1 month, and q/6 months after treatment. There were no clinically significant ocular events; there was no ocular inflammation. The only medication-related change was a separation of the photoreceptor outer segment tip from the apical retinal pigment epithelium that could be traced from the fovea to the perifoveal retina noted in 9/11 (82%) of the patients. There were no changes in retinal pigment epithelium melanization or lipofuscin content by near-infrared fundus autofluorescence and short-wavelength fundus autofluorescence, respectively. There were no inner retinal or outer nuclear layer changes. Visual acuities and sensitivities were unchanged. BRAFi (trametinib) + MEKi (dabrafenib) + HCQ causes very frequent, subclinical separation of the photoreceptor outer segment from the apical retinal pigment epithelium without inner retinal changes or signs of inflammation. The changes suggest interference with the maintenance of the outer retinal barrier and/or phagocytic/pump functions of the retinal pigment epithelium by effective MEK inhibition.

KRAS/BRAF Analysis in Ovarian Low-Grade Serous Carcinoma Having Synchronous All Pathological Precursor Regions

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Kohei Nakamura

2016-04-01

Full Text Available Ovarian low-grade serous carcinoma is thought to begin as a serous cystadenoma or adenofibroma that progresses in a slow stepwise fashion. Among the low-grade serous carcinomas, there is a high frequency of activating mutations in the KRAS or BRAF genes; however, it remains unclear as to how these mutations contribute to tumor progression. This is the first report to track the histopathological progression of serous adenofibroma to low-grade serous carcinoma. Each stage was individually analyzed by pathological and molecular genetic methods to determine what differences occur between the distinct stages of progression.

Clinical significance of BRAF non-V600E mutations on the therapeutic effects of anti-EGFR monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer: the Biomarker Research for anti-EGFR monoclonal Antibodies by Comprehensive Cancer genomics (BREAC) study.

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Shinozaki, Eiji; Yoshino, Takayuki; Yamazaki, Kentaro; Muro, Kei; Yamaguchi, Kensei; Nishina, Tomohiro; Yuki, Satoshi; Shitara, Kohei; Bando, Hideaki; Mimaki, Sachiyo; Nakai, Chikako; Matsushima, Koutatsu; Suzuki, Yutaka; Akagi, Kiwamu; Yamanaka, Takeharu; Nomura, Shogo; Fujii, Satoshi; Esumi, Hiroyasu; Sugiyama, Masaya; Nishida, Nao; Mizokami, Masashi; Koh, Yasuhiro; Abe, Yukiko; Ohtsu, Atsushi; Tsuchihara, Katsuya

2017-11-07

Patients with BRAF V600E -mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAF V600E (BRAF non-V600E mutations) contribute to anti-EGFR antibody resistance. This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort. In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAF V600E (6.0%), and 7 patients with BRAF non-V600E mutations (4.7%), respectively. The response rates in RAS, BRAF V600E , and BRAF non-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAF non-V600E mutations was 2.4 months, similar to that in RAS or BRAF V600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months). Although BRAF non-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAF non-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.

A Retrospective Review of Conjunctival Melanoma Presentation, Treatment, and Outcome and an Investigation of Features Associated With BRAF Mutations

DEFF Research Database (Denmark)

Larsen, Ann-Cathrine; Dahmcke, Christina M.; Dahl, Christina

2015-01-01

IMPORTANCE: Large studies investigating clinical presentation and treatment in primary conjunctival melanoma (CM) are rare. Clinicopathological characteristics of BRAF-mutated CM have not been studied thoroughly. OBJECTIVES: To determine the associations of clinicopathological tumor features...... and treatment with local recurrence, metastasis, and mortality and to determine the association of BRAF mutations with these features. DESIGN, SETTING, AND PARTICIPANTS: Population-based cohort study at the Eye Pathology Institute, Copenhagen, Denmark. Participants included 139 patients with primary CM...

The proto-oncogene KRAS and BRAF profiles and some clinical characteristics in colorectal cancer in the Turkish population.

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Ozen, Filiz; Ozdemir, Semra; Zemheri, Ebru; Hacimuto, Gizem; Silan, Fatma; Ozdemir, Ozturk

2013-02-01

The aim of the current study was to investigate the prevalence and predictive significance of the KRAS and BRAF mutations in Turkish patients with colorectal cancer (CRC). Totally, 53 fresh tumoral tissue specimens were investigated in patients with CRC. All specimens were obtained during routine surgery of patients who were histopathologically diagnosed and genotyped for common KRAS and BRAF point mutations. After DNA extraction, the target mutations were analyzed using the AutoGenomics INFINITI(®) assay, and some samples were confirmed by quantitative real-time polymerase chain reaction fluorescence melting curve analyses. KRAS mutations were found in 26 (49.05%) CRC samples. Twenty-seven samples (50.95%) had wild-type profiles for KRAS codon 12, 13, and 61 in the current cohort. In 17 (65.38%) samples, codon 12; in 7 (26.93%) samples, codon 13; and in 2 (7.69%) samples, codon 61 were found to be mutated, particularly in grade 2 of tumoral tissues. No point mutation was detected in BRAF codon Val600Glu for the studied CRC patients. Our study, based on a representative collection of human CRC tumors, indicates that KRAS gene mutations were detected in 49.05% of the samples, and the most frequent mutation was in the G12D codon. Results also showed that codons 12 and 13 of KRAS are relatively frequently without BRAF mutation in a CRC cohort from the Turkish population.

Activity and Safety of Cetuximab Plus Modified FOLFOXIRI Followed by Maintenance With Cetuximab or Bevacizumab for RAS and BRAF Wild-type Metastatic Colorectal Cancer: A Randomized Phase 2 Clinical Trial.

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Cremolini, Chiara; Antoniotti, Carlotta; Lonardi, Sara; Aprile, Giuseppe; Bergamo, Francesca; Masi, Gianluca; Grande, Roberta; Tonini, Giuseppe; Mescoli, Claudia; Cardellino, Giovanni Gerardo; Coltelli, Luigi; Salvatore, Lisa; Corsi, Domenico Cristiano; Lupi, Cristiana; Gemma, Donatello; Ronzoni, Monica; Dell'Aquila, Emanuela; Marmorino, Federica; Di Fabio, Francesca; Mancini, Maria Laura; Marcucci, Lorenzo; Fontanini, Gabriella; Zagonel, Vittorina; Boni, Luca; Falcone, Alfredo

2018-04-01

The combination of a triple-drug chemotherapy regimen with an anti-epidermal growth factor receptor (EGFR) agent as a first-line treatment of metastatic colorectal cancer (mCRC) showed promising activity along with safety concerns in single-arm phase 2 trials. The role of maintenance following chemotherapy and anti-EGFR and the optimal regimen to be adopted are not established. To evaluate the activity and safety of cetuximab plus modified FOLFOXIRI (mFOLFOXIRI) and explore the role of maintenance with cetuximab or bevacizumab in RAS and BRAF wild-type mCRC. In a prospective, noncomparative, open-label, multicenter, randomized phase 2 trial, patients aged 18 to 75 years with unresectable, previously untreated RAS and BRAF wild-type (before amendment, KRAS wild-type) mCRC were recruited from 21 oncology units in Italy from October 19, 2011, to March 1, 2015 (followed up through May 31, 2017). In total, 323 patients were screened and 143 were randomized to 2 treatment arms to receive as a first-line induction a regimen of mFOLFOXIRI plus cetuximab followed by cetuximab (arm A) or bevacizumab (arm B) until disease progression. Primary analyses were conducted in a modified intention-to-treat population. mFOLFOXIRI plus cetuximab repeated every 2 weeks for up to 8 cycles, followed by maintenance with cetuximab or bevacizumab until disease progression. The primary end point was the 10-month progression-free rate (PFR); secondary end points included progression-free and overall survival, response rate, rate of metastases resection, and adverse events. Of 143 patients randomized, 116 (81.1%) (median [interquartile range (IQR)] age, 59.5 [53-67] years; 34 [29.3%] women) had RAS and BRAF wild-type mCRC. At a median (IQR) follow-up of 44.0 (30.5-52.1) months, 10-month PFRs were 50.8% (90% CI, 39.5%-62.2%) in arm A and 40.4% (90% CI, 29.4%-52.1%) in arm B. The overall response rate was 71.6% (95% CI, 62.4%-79.5%). Main grade 3/4 adverse events were neutropenia (occurring in 36

BRAF and MEK Inhibitors Influence the Function of Reprogrammed T Cells: Consequences for Adoptive T-Cell Therapy

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Jan Dörrie

2018-01-01

Full Text Available BRAF and MEK inhibitors (BRAFi/MEKi, the standard treatment for patients with BRAFV600 mutated melanoma, are currently explored in combination with various immunotherapies, notably checkpoint inhibitors and adoptive transfer of receptor-transfected T cells. Since two BRAFi/MEKi combinations with similar efficacy are approved, potential differences in their effects on immune cells would enable a rational choice for triple therapies. Therefore, we characterized the influence of the clinically approved BRAFi/MEKi combinations dabrafenib (Dabra and trametinib (Tram vs. vemurafenib (Vem and cobimetinib (Cobi on the activation and functionality of chimeric antigen receptor (CAR-transfected T cells. We co-cultured CAR-transfected CD8+ T cells and target cells with clinically relevant concentrations of the inhibitors and determined the antigen-induced cytokine secretion. All BRAFi/MEKi reduced this release as single agents, with Dabra having the mildest inhibitory effect, and Dabra + Tram having a clearly milder inhibitory effect than Vem + Cobi. A similar picture was observed for the upregulation of the activation markers CD25 and CD69 on CAR-transfected T cells after antigen-specific stimulation. Most importantly, the cytolytic capacity of the CAR-T cells was significantly inhibited by Cobi and Vem + Cobi, whereas the other kinase inhibitors showed no effect. Therefore, the combination Dabra + Tram would be more suitable for combining with T-cell-based immunotherapy than Vem + Cobi.

Systemic retinoids for the chemoprevention of cutaneous squamous cell carcinoma and verrucal keratosis in a cohort of patients on BRAF inhibitors.

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Anforth, R; Blumetti, T C M P; Clements, A; Kefford, R; Long, G V; Fernandez-Peñas, P

2013-12-01

The treatment of metastatic melanoma has changed greatly with the development of inhibitors targeted at the mutated BRAF kinase present in up to 50% of metastatic melanoma cases. These agents, vemurafenib and dabrafenib, have been shown to increase median survival. Unfortunately, they have also been associated with the development of verrucal keratosis (VK) and cutaneous squamous cell carcinoma (cuSCC). These lesions require surgical excision, and when a large number of these lesions need to be treated, it can significantly affect the patient's quality of life. To determine if acitretin is suitable as a chemopreventative agent against the development of verrucal keratosis and cuSCC, in patients on BRAF inhibitors. Patients treated with a BRAF inhibitor, vemurafenib or dabrafenib, for stage IV metastatic melanoma, who had undergone more than five surgical excisions to remove lesions suggestive of cuSCC, were offered the opportunity to commence acitretin as a chemopreventative agent. Patients were evaluated every 4 weeks. Clinical and histological data were collected. Eight patients, who had a total of 24 cuSCC removed, were included in the study. After commencement of acitretin, only five cuSCC were excised from two patients. The most significant reduction was in a patient who had developed 13 cuSCC over 10 months and only two cuSCC 3 months after commencing acitretin. No modifications in the dose of the BRAF inhibitor were made as a result of cuSCC in any of these patients. Acitretin should be considered as a chemopreventative agent for VK and cuSCC in patients taking BRAF inhibitors, before considering dosage reductions. © 2013 British Association of Dermatologists.

Location of Primary Tumor and Benefit From Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies in Patients With RAS and BRAF Wild-Type Metastatic Colorectal Cancer

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Moretto, Roberto; Cremolini, Chiara; Rossini, Daniele; Pietrantonio, Filippo; Battaglin, Francesca; Mennitto, Alessia; Bergamo, Francesca; Loupakis, Fotios; Marmorino, Federica; Berenato, Rosa; Marsico, Valentina Angela; Caporale, Marta; Antoniotti, Carlotta; Masi, Gianluca; Salvatore, Lisa; Borelli, Beatrice; Fontanini, Gabriella; Lonardi, Sara; De Braud, Filippo

2016-01-01

Introduction. Right- and left-sided colorectal cancers (CRCs) differ in clinical and molecular characteristics. Some retrospective analyses suggested that patients with right-sided tumors derive less benefit from anti-epidermal growth factor receptor (EGFR) antibodies; however, molecular selection in those studies was not extensive. Patients and Methods. Patients with RAS and BRAF wild-type metastatic CRC (mCRC) who were treated with single-agent anti-EGFRs or with cetuximab-irinotecan (if refractory to previous irinotecan) were included in the study. Differences in outcome between patients with right- and left-sided tumors were investigated. Results. Of 75 patients, 14 and 61 had right- and left-sided tumors, respectively. None of the right-sided tumors responded according to RECIST, compared with 24 left-sided tumors (overall response rate: 0% vs. 41%; p = .0032), and only 2 patients with right-sided tumors (15%) versus 47 patients with left-sided tumors (80%) achieved disease control (p < .0001). The median duration of progression-free survival was 2.3 and 6.6 months in patients with right-sided and left-sided tumors, respectively (hazard ratio: 3.97; 95% confidence interval: 2.09–7.53; p < .0001). Conclusion. Patients with right-sided RAS and BRAF wild-type mCRC seemed to derive no benefit from single-agent anti-EGFRs. Implications for Practice: Right- and left-sided colorectal tumors have peculiar epidemiological and clinicopathological characteristics, distinct gene expression profiles and genetic alterations, and different prognoses. This study assessed the potential predictive impact of primary tumor site with regard to anti-epidermal growth factor receptor (EGFR) monoclonal antibody treatment in patients with RAS and BRAF wild-type metastatic colorectal cancer. The results demonstrated the lack of activity of anti-EGFRs in RAS and BRAF wild-type, right-sided tumors, thus suggesting a potential role for primary tumor location in driving treatment choices

Recurrent papillary craniopharyngioma with BRAF V600E mutation treated with dabrafenib: case report.

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Himes, Benjamin T; Ruff, Michael W; Van Gompel, Jaimie J; Park, Sean S; Galanis, Evanthia; Kaufmann, Timothy J; Uhm, Joon H

2018-04-27

The authors present the case of a man with a papillary craniopharyngioma, first diagnosed at 47 years of age, who experienced multiple recurrences. Review of the pathologic specimen from his first resection demonstrated the BRAF V600E mutation. With his most recent recurrence following previous surgery and radiotherapy, at 52 years of age, the decision was made to initiate treatment with the BRAF V600E inhibitor dabrafenib. Imaging following initiation of dabrafenib demonstrated reduction in tumor size. He remained on dabrafenib therapy for approximately 1 year and continued to demonstrate a good clinical result. At that time the decision was made to discontinue dabrafenib therapy and follow up with serial imaging. After more than 1 year of follow-up since stopping dabrafenib, the patient has continued to do well with no radiographic evidence of tumor progression and continues to be monitored with frequent interval imaging.

Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

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Zecena, Helma; Tveit, Daniel; Wang, Zi; Farhat, Ahmed; Panchal, Parvita; Liu, Jing; Singh, Simar J; Sanghera, Amandeep; Bainiwal, Ajay; Teo, Shuan Y; Meyskens, Frank L; Liu-Smith, Feng; Filipp, Fabian V

2018-04-04

Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance

Phosphoproteomic analysis of cell-based resistance to BRAF inhibitor therapy in melanoma

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Robert eParker

2015-05-01

Full Text Available The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition in vitro. A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, Protein Kinase C, IGF signaling and melanosome maturation were highly divergent after transition to a drug resistant phenotype.

A novel approach to detect KRAS/BRAF mutation for colon cancer: Highly sensitive simultaneous detection of mutations and simple pre-treatment without DNA extraction.

Science.gov (United States)

Suzuki, Shun-Ichi; Matsusaka, Satoshi; Hirai, Mitsuharu; Shibata, Harumi; Takagi, Koichi; Mizunuma, Nobuyuki; Hatake, Kiyohiko

2015-07-01

It has been reported that colon cancer patients with KRAS and BRAF mutations that lie downstream of epidermal growth factor receptor (EGFR) acquire resistance against therapy with anti‑EGFR antibodies, cetuximab and panitumumab. On the other hand, some reports say KRAS codon 13 mutation (p.G13D) has lower resistance against anti-EGFR antibodies, thus there is a substantial need for detection of specific KRAS mutations. We have established a state-of-the-art measurement system using QProbe (QP) method that allows simultaneous measurement of KRAS codon 12/13, p.G13D and BRAF mutation, and compared this method against Direct Sequencing (DS) using 182 specimens from colon cancer patients. In addition, 32 biopsy specimens were processed with a novel pre-treatment method without DNA purification in order to detect KRAS/BRAF. As a result of KRAS mutation measurement, concordance rate between the QP method and DS method was 81.4% (144/177) except for the 5 specimens that were undeterminable. Among them, 29 specimens became positive with QP method and negative with DS method. BRAF was measured with QP method only, and the mutation detection rate was 3.9% (6/153). KRAS measurement using a simple new pre-treatment method without DNA extraction resulted in 31 good results out of 32, all of them matching with the DS method. We have established a simple but highly sensitive simultaneous detection system for KRAS/BRAF. Moreover, introduction of the novel pre-treatment technology eliminated the inconvenient DNA extraction process. From this research achievement, we not only anticipate quick and accurate results returned in the clinical field but also contribution in improving the test quality and work efficiency.

First-in-Man Dose-Escalation Study of the Selective BRAF Inhibitor RG7256 in Patients with BRAF V600-Mutated Advanced Solid Tumors

DEFF Research Database (Denmark)

Dienstmann, Rodrigo; Lassen, Ulrik; Cebon, Jonathan

2016-01-01

V600-mutated advanced solid tumors. PATIENTS AND METHODS: Patients received RG7256 orally over 8 dose levels from 200 mg once a day (QD) to 2400 mg twice a day (BID) (50-, 100- and 150-mg tablets) using a classic 3 + 3 dose escalation design. RESULTS: In total, 45 patients were enrolled; most (87...... %) had advanced melanoma (94 % BRAF V600E). RG7256 was rapidly absorbed, with limited accumulation and dose-proportional increase in exposure up to 1950 mg BID. The maximal tolerated dose (MTD) was not reached. The most common drug-related adverse events (AEs) were dyspepsia (20 %), dry skin (18 %), rash...

Are All Mutations the Same? A Rare Case Report of Coexisting Mutually Exclusive KRAS and BRAF Mutations in a Patient with Metastatic Colon Adenocarcinoma

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Anusha Vittal

2017-01-01

Full Text Available 29-year-old Hispanic woman presented to the clinic with complaints of abdominal pain, nausea, fatigue, and constipation. Laboratory tests indicated the presence of iron deficiency anemia and transaminitis. Imaging evaluation revealed marked hepatomegaly with multiple hepatic metastases and pelvic lymphadenopathy. Biopsy of the hepatic lesions showed adenocarcinoma positive for pan-cytokeratin, CMA5.2, villin, and CDX2. She was positive for tumor markers CA 19-9, CA-125, and CEA. Upon further evaluation, she was found to have colorectal cancer positive for KRAS and BRAF mutations. Unfortunately, her disease progressed rapidly and she expired within 3 months from the time of her first diagnosis. KRAS and BRAF mutations are rare enough to be considered virtually mutually exclusive but coexistent mutations appear to be a distinct molecular and clinical subset with aggressive course of illness, which is in dire need of new treatment strategies. Panitumumab and Cetuximab are approved for patients with wild type KRAS CRC. Vemurafenib is a potent inhibitor of the kinase domain in mutant BRAF and its use in BRAF mutated colon cancer remains to be well established. Our report highlights the need to obtain tissue samples from these patients for analysis and to evaluate the benefit of Vemurafenib in colorectal cancers.

Dabrafenib in patients with Val600Glu or Val600Lys BRAF-mutant melanoma metastatic to the brain (BREAK-MB): a multicentre, open-label, phase 2 trial.

Science.gov (United States)

Long, Georgina V; Trefzer, Uwe; Davies, Michael A; Kefford, Richard F; Ascierto, Paolo A; Chapman, Paul B; Puzanov, Igor; Hauschild, Axel; Robert, Caroline; Algazi, Alain; Mortier, Laurent; Tawbi, Hussein; Wilhelm, Tabea; Zimmer, Lisa; Switzky, Julie; Swann, Suzanne; Martin, Anne-Marie; Guckert, Mary; Goodman, Vicki; Streit, Michael; Kirkwood, John M; Schadendorf, Dirk

2012-11-01

-related adverse events of grade 3 or worse occurred in 38 (22%) patients. Eleven (6%) patients developed squamous-cell carcinoma (five [6%] patients in cohort A, of whom one also had keratoacanthoma; six [7%] in cohort B). Four grade 4 treatment-related adverse events occurred in cohort A: one blood amylase increase, one convulsion, one lipase increase, and one neutropenia. Two grade 4 events occurred in cohort B: one agranulocytosis and one intracranial haemorrhage. 51 (30%) patients had a serious adverse event. The three most frequent serious adverse events were pyrexia (ten [6%] patients), intracranial haemorrhage (ten [6%]; one treatment-related), and squamous-cell carcinoma (11 [6%]). Dabrafenib has activity and an acceptable safety profile in patients with Val600Glu BRAF-mutant melanoma and brain metastases irrespective of whether they are untreated or have been previously treated but have progressed. GlaxoSmithKline. Copyright © 2012 Elsevier Ltd. All rights reserved.

Exploiting cannabinoid-induced cytotoxic autophagy to drive melanoma cell death.

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Armstrong, Jane L; Hill, David S; McKee, Christopher S; Hernandez-Tiedra, Sonia; Lorente, Mar; Lopez-Valero, Israel; Eleni Anagnostou, Maria; Babatunde, Fiyinfoluwa; Corazzari, Marco; Redfern, Christopher P F; Velasco, Guillermo; Lovat, Penny E

2015-06-01

Although the global incidence of cutaneous melanoma is increasing, survival rates for patients with metastatic disease remain viability, and activation of apoptosis, whereas cotreatment with chloroquine or knockdown of Atg7, but not Beclin-1 or Ambra1, prevented THC-induced autophagy and cell death in vitro. Administration of Sativex-like (a laboratory preparation comprising equal amounts of THC and cannabidiol (CBD)) to mice bearing BRAF wild-type melanoma xenografts substantially inhibited melanoma viability, proliferation, and tumor growth paralleled by an increase in autophagy and apoptosis compared with standard single-agent temozolomide. Collectively, our findings suggest that THC activates noncanonical autophagy-mediated apoptosis of melanoma cells, suggesting that cytotoxic autophagy induction with Sativex warrants clinical evaluation for metastatic disease.

Impact of KRAS, BRAF, PIK3CA mutations, PTEN, AREG, EREG expression and skin rash in ≥ 2 line cetuximab-based therapy of colorectal cancer patients.

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Zacharenia Saridaki

2011-01-01

Full Text Available To investigate the predictive significance of KRAS, BRAF, PIK3CA mutational status, AREG- EREG mRNA expression, PTEN protein expression and skin rash in metastatic colorectal cancer (mCRC patients treated with cetuximab containing salvage chemotherapy.Primary tumors from 112 mCRC patients were analyzed. The worst skin toxicity during treatment was recorded.KRAS, BRAF and PIK3CA mutations were present in 37 (33%, 8 (7.2% and 11 (9.8% cases, respectively, PTEN was lost in 21 (19.8% cases, AREG and EREG were overexpressed in 48 (45% and 51 (49% cases. In the whole study population, time to tumor progression (TTP and overall survival (OS was significantly lower in patients with KRAS (p = 0.001 and p = 0.026, respectively or BRAF (p = 0.001 and p<0.0001, respectively mutant tumors, downregulation of AREG (p = 0.018 and p = 0.013, respectively or EREG (p = 0.002 and p = 0.004, respectively and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively. In KRAS wt patients TTP and OS was significantly lower in patients with BRAF (p = 0.0001 and p<0.0001, respectively mutant tumors, downregulation of AREG (p = 0.021 and p = 0.004, respectively or EREG (p = 0.0001 and p<0.0001, respectively and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively. TTP was significantly lower in patients with PIK3CA mutations (p = 0.01 or lost PTEN (p = 0.002. Multivariate analysis revealed KRAS (Hazard Ratio [HR] 4.3, p<0.0001, BRAF mutation (HR: 5.1, p<0.0001, EREG low expression (HR: 1.6, p = 0.021 and absence of severe/moderate skin rash (HR: 4.0, p<0.0001 as independent prognostic factors for decreased TTP. Similarly, KRAS (HR 2.9, p = 0.01, BRAF mutation (HR: 3.0, p = 0.001, EREG low expression (HR: 1.7, p = 0.021, absence of severe/moderate skin rash (HR: 3.7, p<0.0001 and the presence of undifferantited tumours (HR: 2.2, p = 0.001 were revealed as independent prognostic factors for decreased OS.These results underscore that KRAS-BRAF mutations and EREG

Erdheim Chester disease with appendicular skeletal, renal and pleural involvement responding to Zelboraf (BRAF inhibitor) treatment: case report

International Nuclear Information System (INIS)

Borys, Dariusz; Nystrom, Lucas; Song, Albert; Lomasney, Laurie M.

2016-01-01

Erdheim Chester disease is a rare non-Langerhans cell histiocytosis which may involve multiple organs including bone, soft tissue, lungs, cardiovascular system, kidneys (retroperitoneum), skin, and central nervous system. Bone involvement is most common followed by other organs. This case report describes a 58-year-old man who presented with progressive renal dysfunction presumed due to obstruction. The patient failed multiple urinary tract interventions, and clinical course was complicated by recurrent low-grade fevers, and bilateral knee pain. Advanced imaging and histopathological features on bone biopsy were consistent with Erdheim Chester disease. Molecular studies of tissue showed BRAF V600 mutation. This patient was treated with Zelboraf (vemurafenib) BRAF inhibitor with subsequent improvement in renal and pleural dysfunction as well as decreased histiocytic soft tissue masses on CT. (orig.)

Erdheim Chester disease with appendicular skeletal, renal and pleural involvement responding to Zelboraf (BRAF inhibitor) treatment: case report

Energy Technology Data Exchange (ETDEWEB)

Borys, Dariusz [Loyola University Medical Center Chicago, Department of Pathology, Maywood, IL (United States); Loyola University Medical Center Chicago, Departmet of Orthopaedic Surgery, Maywood, IL (United States); Loyola University Medical Center, Maywood, IL (United States); Nystrom, Lucas [Loyola University Medical Center Chicago, Departmet of Orthopaedic Surgery, Maywood, IL (United States); Song, Albert [Loyola University Medical Center Chicago, Department of Radiology, Maywood, IL (United States); Lomasney, Laurie M. [Loyola University Medical Center Chicago, Departmet of Orthopaedic Surgery, Maywood, IL (United States); Loyola University Medical Center Chicago, Department of Radiology, Maywood, IL (United States)

2016-10-15

Erdheim Chester disease is a rare non-Langerhans cell histiocytosis which may involve multiple organs including bone, soft tissue, lungs, cardiovascular system, kidneys (retroperitoneum), skin, and central nervous system. Bone involvement is most common followed by other organs. This case report describes a 58-year-old man who presented with progressive renal dysfunction presumed due to obstruction. The patient failed multiple urinary tract interventions, and clinical course was complicated by recurrent low-grade fevers, and bilateral knee pain. Advanced imaging and histopathological features on bone biopsy were consistent with Erdheim Chester disease. Molecular studies of tissue showed BRAF V600 mutation. This patient was treated with Zelboraf (vemurafenib) BRAF inhibitor with subsequent improvement in renal and pleural dysfunction as well as decreased histiocytic soft tissue masses on CT. (orig.)

Targeting BRAF V600E and Autophagy in Pediatric Brain Tumors

Science.gov (United States)

2015-10-01

tumors in vivo by using combination therapy with chloroquine (autophagy inhibition) and vemurafenib (Raf-inhibitor). The major goals of the project as...BRAFV600E Cell lines used : BRafCA/WTInk4alox/lox mouse neurospheres (Cell line provided by Dr. Claudia Petritsch, UCSF). We have been able to successfully...a dose dependent manner to chloroquine as shown by MTS and LDH. 0 1.25 2.5 5 10 20 40 0 50 100 150 CQ (µM) % S ur vi va l 0 1 2 3 LD H release

Intra-tumoral Heterogeneity of KRAS and BRAF Mutation Status in Patients with Advanced Colorectal Cancer (aCRC and Cost-Effectiveness of Multiple Sample Testing

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Susan D. Richman

2011-01-01

Full Text Available KRAS mutation status is established as a predictive biomarker of benefit from anti-EGFr therapies. Mutations are normally assessed using DNA extracted from one formalin-fixed, paraffin-embedded (FFPE tumor block. We assessed heterogeneity of KRAS and BRAF mutation status intra-tumorally (multiple blocks from the same primary tumor. We also investigated the utility and efficiency of genotyping a ‘DNA cocktail’ prepared from multiple blocks. We studied 68 consenting patients in two randomized clinical trials. DNA was extracted, from ≥2 primary tumor FFPE blocks per patient. DNA was genotyped by pyrosequencing for KRAS codons 12, 13 and 61 and BRAF codon 600. In patients with heterogeneous mutation status, DNA cocktails were prepared and genotyped. Among 69 primary tumors in 68 patients, 7 (10.1% showed intratumoral heterogeneity; 5 (7.2% at KRAS codons 12, 13 and 2 (2.9% at BRAF codon 600. In patients displaying heterogeneity, the relevant KRAS or BRAF mutation was also identified in ‘DNA cocktail’ samples when including DNA from mutant and wild-type blocks. Heterogeneity is uncommon but not insignificant. Testing DNA from a single block will wrongly assign wild-type status to 10% patients. Testing more than one block, or preferably preparation of a ‘DNA cocktail’ from two or more tumor blocks, improves mutation detection at minimal extra cost.

TRAIL receptor upregulation and the implication of KRAS/BRAF mutations in human colon cancer tumours

Czech Academy of Sciences Publication Activity Database

Oikonomou, E.; Kosmidou, V.; Katseli, A.; Kothonidis, K.; Mourtzoukou, D.; Kontogeorgos, G.; Anděra, Ladislav; Zografos, G.; Pintzas, A.

2009-01-01

Roč. 125, č. 9 (2009), s. 2127-2135 ISSN 0020-7136 R&D Projects: GA MŠk 1M0506 Grant - others:EC(XE) LSHC-CT-2006-037278 Institutional research plan: CEZ:AV0Z50520514 Keywords : colorectal tumours * TRAIL receptors expression * KRAS/ BRAF oncogenic mutations Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.722, year: 2009

Clinical profiling of BCL-2 family members in the setting of BRAF inhibition offers a rationale for targeting de novo resistance using BH3 mimetics.

Directory of Open Access Journals (Sweden)

Dennie T Frederick

Full Text Available While response rates to BRAF inhibitiors (BRAFi are high, disease progression emerges quickly. One strategy to delay the onset of resistance is to target anti-apoptotic proteins such as BCL-2, known to be associated with a poor prognosis. We analyzed BCL-2 family member expression levels of 34 samples from 17 patients collected before and 10 to 14 days after treatment initiation with either vemurafenib or dabrafenib/trametinib combination. The observed changes in mRNA and protein levels with BRAFi treatment led us to hypothesize that combining BRAFi with a BCL-2 inhibitor (the BH3-mimetic navitoclax would improve outcome. We tested this hypothesis in cell lines and in mice. Pretreatment mRNA levels of BCL-2 negatively correlated with maximal tumor regression. Early increases in mRNA levels were seen in BIM, BCL-XL, BID and BCL2-W, as were decreases in MCL-1 and BCL2A. No significant changes were observed with BCL-2. Using reverse phase protein array (RPPA, significant increases in protein levels were found in BIM and BID. No changes in mRNA or protein correlated with response. Concurrent BRAF (PLX4720 and BCL2 (navitoclax inhibition synergistically reduced viability in BRAF mutant cell lines and correlated with down-modulation of MCL-1 and BIM induction after PLX4720 treatment. In xenograft models, navitoclax enhanced the efficacy of PLX4720. The combination of a selective BRAF inhibitor with a BH3-mimetic promises to be an important therapeutic strategy capable of enhancing the clinical efficacy of BRAF inhibition in many patients that might otherwise succumb quickly to de novo resistance. Trial registrations: ClinicalTrials.gov NCT01006980; ClinicalTrials.gov NCT01107418; ClinicalTrials.gov NCT01264380; ClinicalTrials.gov NCT01248936; ClinicalTrials.gov NCT00949702; ClinicalTrials.gov NCT01072175.

The role of APC in WNT pathway activation in serrated neoplasia.

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Borowsky, Jennifer; Dumenil, Troy; Bettington, Mark; Pearson, Sally-Ann; Bond, Catherine; Fennell, Lochlan; Liu, Cheng; McKeone, Diane; Rosty, Christophe; Brown, Ian; Walker, Neal; Leggett, Barbara; Whitehall, Vicki

2018-03-01

Conventional adenomas are initiated by APC gene mutation that activates the WNT signal. Serrated neoplasia is commonly initiated by BRAF or KRAS mutation. WNT pathway activation may also occur, however, to what extent this is owing to APC mutation is unknown. We examined aberrant nuclear β-catenin immunolocalization as a surrogate for WNT pathway activation and analyzed the entire APC gene coding sequence in serrated and conventional pathway polyps and cancers. WNT pathway activation was a common event in conventional pathway lesions with aberrant nuclear immunolocalization of β-catenin and truncating APC mutations in 90% and 89% of conventional adenomas and 82% and 70% of BRAF wild-type cancers, respectively. WNT pathway activation was seen to a lesser extent in serrated pathway lesions. It occurred at the transition to dysplasia in serrated polyps with a significant increase in nuclear β-catenin labeling from sessile serrated adenomas (10%) to sessile serrated adenomas with dysplasia (55%) and traditional serrated adenomas (9%) to traditional serrated adenomas with dysplasia (39%) (P=0.0001). However, unlike the conventional pathway, truncating APC mutations were rare in the serrated pathway lesions especially sessile serrated adenomas even when dysplastic (15%) and in the BRAF mutant cancers with microsatellite instability that arise from them (8%). In contrast, APC missense mutations that were rare in conventional pathway adenomas and cancers (3% in BRAF wild-type cancers) were more frequent in BRAF mutant cancers with microsatellite instability (32%). We conclude that increased WNT signaling is important in the transition to malignancy in the serrated pathway but that APC mutation is less common and the spectrum of mutations is different than in conventional colorectal carcinogenesis. Moderate impact APC mutations and non-APC-related causes of increased WNT signaling may have a more important role in serrated neoplasia than the truncating APC mutations

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

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Wu, Xue; Yin, Jiani; Simpson, Jeremy; Kim, Kyoung-Han; Gu, Shengqing; Hong, Jenny H.; Bayliss, Peter; Backx, Peter H.

2012-01-01

Noonan syndrome (NS) is a relatively common autosomal dominant disorder characterized by congenital heart defects, short stature, and facial dysmorphia. NS is caused by germ line mutations in several components of the RAS–RAF–MEK–extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, including both kinase-activating and kinase-impaired alleles of RAF1 (∼3 to 5%), which encodes a serine-threonine kinase for MEK1/2. To investigate how kinase-impaired RAF1 mutants cause NS, we generated knock-in mice expressing Raf1D486N. Raf1D486N/+ (here D486N/+) female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1D486N-expressing cells compared with controls. RAF1D486N, as well as other kinase-impaired RAF1 mutants, showed increased heterodimerization with BRAF, which was necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also required heterodimerization to enhance MEK/ERK activation. Our results suggest that an increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations. PMID:22826437

IAP antagonists Birinapant and AT-406 efficiently synergise with either TRAIL, BRAF, or BCL-2 inhibitors to sensitise BRAFV600E colorectal tumour cells to apoptosis.

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Perimenis, Philippos; Galaris, Apostolos; Voulgari, Alexandra; Prassa, Margarita; Pintzas, Alexander

2016-08-12

High expression levels of Inhibitors of Apoptosis Proteins (IAPs) have been correlated with poor cancer prognosis and block the cell death pathway by interfering with caspase activation. SMAC-mimetics are small-molecule inhibitors of IAPs that mimic the endogenous SMAC and promote the induction of cell death by neutralizing IAPs. In this study, anti-tumour activity of new SMAC-mimetics Birinapant and AT-406 is evaluated against colorectal adenocarcinoma cells and IAP cross-talk with either oncogenic BRAF or BCL-2, or with the TRAIL are further exploited towards rational combined protocols. It is shown that pre-treatment of SMAC-mimetics followed by their combined treatment with BRAF inhibitors can decrease cell viability, migration and can very efficiently sensitize colorectal tumour cells to apoptosis. Moreover, co-treatment of TRAIL with SMAC-mimetics can efficiently sensitize resistant tumour cells to apoptosis synergistically, as shown by median effect analysis. Finally, Birinapant and AT-406 can synergise with BCL-2 inhibitor ABT-199 to reduce viability of adenocarcinoma cells with high BCL-2 expression. Proposed synergistic rational anticancer combined protocols of IAP antagonists Birinapant and AT-406 in 2D and 3D cultures can be later further exploited in vivo, from precision tumour biology to precision medical oncology.

Endometrial tumour BRAF mutations and MLH1 promoter methylation as predictors of germline mismatch repair gene mutation status: a literature review.

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Metcalf, Alexander M; Spurdle, Amanda B

2014-03-01

Colorectal cancer (CRC) that displays high microsatellite instability (MSI-H) can be caused by either germline mutations in mismatch repair (MMR) genes, or non-inherited transcriptional silencing of the MLH1 promoter. A correlation between MLH1 promoter methylation, specifically the 'C' region, and BRAF V600E status has been reported in CRC studies. Germline MMR mutations also greatly increase risk of endometrial cancer (EC), but no systematic review has been undertaken to determine if these tumour markers may be useful predictors of MMR mutation status in EC patients. Endometrial cancer cohorts meeting review inclusion criteria encompassed 2675 tumours from 20 studies for BRAF V600E, and 447 tumours from 11 studies for MLH1 methylation testing. BRAF V600E mutations were reported in 4/2675 (0.1%) endometrial tumours of unknown MMR mutation status, and there were 7/823 (0.9%) total sequence variants in exon 11 and 27/1012 (2.7%) in exon 15. Promoter MLH1 methylation was not observed in tumours from 32 MLH1 mutation carriers, or for 13 MSH2 or MSH6 mutation carriers. MMR mutation-negative individuals with tumour MLH1 and PMS2 IHC loss displayed MLH1 methylation in 48/51 (94%) of tumours. We have also detailed specific examples that show the importance of MLH1 promoter region, assay design, and quantification of methylation. This review shows that BRAF mutations occurs so infrequently in endometrial tumours they can be discounted as a useful marker for predicting MMR-negative mutation status, and further studies of endometrial cohorts with known MMR mutation status are necessary to quantify the utility of tumour MLH1 promoter methylation as a marker of negative germline MMR mutation status in EC patients.

Genetic study of the BRAF gene reveals new variants and high frequency of the V600E mutation among Iranian ameloblastoma patients.

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Soltani, Maryam; Tabatabaiefar, Mohammad Amin; Mohsenifar, Zhaleh; Pourreza, Mohammad Reza; Moridnia, Abbas; Shariati, Laleh; Razavi, Seyyed Mohammad

2018-01-01

Ameloblastoma is a benign, slow-growing and locally invasive tumor. It is one of the most prevalent odontogenic tumors, with an incidence rate of 1% of all oral tumors and approximately 18% of odontogenic tumors. A group of genes have been investigated in patients with ameloblastoma. The BRAF V600E mutation has been implicated as the most common mutation in ameloblastoma. The presence or absence of this mutation has been associated with several clinicopathological properties, including location, age at diagnosis, histology, and prognosis. Although some populations have been investigated so far, little data are available on the Iranian population. The current research was launched to study the BRAF V600E mutation among a cohort of Iranian patients with ameloblastoma. In this clinicopathological and molecular biology study, a total of 19 formalin-fixed, paraffin-embedded tissues were studied. DNA extraction was performed, followed by PCR-sequencing of exons 10 and 15 of the BRAF gene to identify mutations. In silico analysis was performed for the identified variants. Results were analyzed by T test, Chi-square, and Fisher's exact test. Totally, 12 of 19 samples (63%) harbored the p. V600E hotspot mutation. In addition, we identified several variants, two of which were novel. The c.1769T>G (p. V590G) and c.1751C>T (p.L584F) as the novel variants showed a possible damaging effect by in silico analysis. No variant was found within exon 10. Our study confirms the role of BRAF mutations in ameloblastoma in the Iranian patients studied. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Durable complete responses off all treatment in patients with metastatic malignant melanoma after sequential immunotherapy followed by a finite course of BRAF inhibitor therapy.

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Wyluda, Edward J; Cheng, Jihua; Schell, Todd D; Haley, Jeremy S; Mallon, Carol; Neves, Rogerio I; Robertson, Gavin; Sivik, Jeffrey; Mackley, Heath; Talamo, Giampaolo; Drabick, Joseph J

2015-01-01

We report 3 cases of durable complete response (CR) in patients with BRAF-mutated metastatic melanoma who were initially treated unsuccessfully with sequential immunotherapies (high dose interleukin 2 followed by ipilimumab with or without concurrent radiation therapy). After progression during or post immunotherapy, these patients were given BRAF inhibitor therapy and developed rapid CRs. Based on the concomitant presence of autoimmune manifestations (including vitiligo and hypophysitis), we postulated that there was a synergistic effect between the prior immune therapy and the BRAF targeting agents. Accordingly, the inhibitors were gradually weaned off beginning at 3 months and were stopped completely at 9-12 months. The three patients remain well and in CR off of all therapy at up to 15 months radiographic follow-up. The institution of the BRAF therapy was associated with development of severe rheumatoid-like arthritis in 2 patients which persisted for months after discontinuation of therapy, suggesting it was not merely a known toxicity of BRAF inhibitors (arthralgias). On immunologic analysis, these patients had high levels of non-T-regulatory, CD4 positive effector phenotype T-cells, which persisted after completion of therapy. Of note, we had previously reported a similar phenomenon in patients with metastatic melanoma who failed high dose interleukin-2 and were then placed on a finite course of temozolomide with rapid complete responses that have remained durable for many years after discontinuation of temozolomide. We postulate that a finite course of cytotoxic or targeted therapy specific for melanoma given after apparent failure of prior immunotherapy can result in complete and durable remissions that may persist long after the specific cytotoxic or targeted agents have been discontinued suggesting the existence of sequence specific synergism between immunotherapy and these agents. Here, we discuss these cases in the context of the literature on

EGFR, ALK, RET, KRAS and BRAF alterations in never-smokers with non-small cell lung cancer.

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Dong, Y U; Ren, Weihong; Qi, Jun; Jin, B O; Li, Ying; Tao, Huiqing; Xu, Ren; Li, Yanqing; Zhang, Qinxian; Han, Baohui

2016-04-01

Non-small cell lung cancer (NSCLC), caused by various mutations in a spectrum of cancer driver genes, may have distinct pathological characteristics and drug responses. Extensive genetic screening and pathological characterization is required for the design of customized therapies to improve patient outcomes. Notably, NSCLC in never-smokers exhibits distinctive clinicopathological features, which are frequently associated with tumorigenic mutations, and thus may be treated as a unique disease entity. However, to the best of our knowledge, these mutations have not been extensively and accurately characterized in an NSCLC study with a large sample size. Therefore, the present study enrolled a large cohort of NSCLC patients, which consisted of 358 never-smokers, for the screening of genetic alterations in the epidermal growth factor receptor (EGFR), ret proto-oncogene (RET), anaplastic lymphoma kinase (ALK), Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-Raf proto-oncogene serine/threonine kinase (BRAF) tumorigenic genes. It was identified that the mutation rate was 47.8, 7.5, 3.6, 1.4 and 0.3% for EGFR, ALK, KRAS, RET and BRAF, respectively. In addition, clinicopathological features associated with these mutations were characterized. EGFR mutations were more frequently observed in female and older patients. By contrast, KRAS mutations were more frequently detected in male patients, and ALK and RET translocations in younger patients. The cancer cells were frequently well-differentiated in carcinoma cases exhibiting EGFR mutations, however, were less differentiated in those with ALK translocations. In conclusion, the present study determined the frequency of oncogenic alterations and associated clinicopathological features in NSCLC exhibited by never-smokers using a large sample size. The results of the present study may enrich our knowledge of NSCLC in never-smokers and provide useful insights for improvement of the outcome of molecularly targeted therapies

Prognostic value of BRAF and KRAS mutation status in stage II and III microsatellite instable colon cancers

NARCIS (Netherlands)

de Cuba, E. M. V.; Snaebjornsson, P.; Heideman, D. A. M.; van Grieken, N. C. T.; Bosch, L. J. W.; Fijneman, R. J. A.; Belt, E.; Bril, H.; Stockmann, H. B. A. C.; Hooijberg, E.; Punt, C. J. A.; Koopman, M.; Nagtegaal, I. D.; Coupé, V. H. M.; Carvalho, B.; Meijer, G. A.

2016-01-01

Microsatellite instability (MSI) has been associated with favourable survival in early stage colorectal cancer (CRC) compared to microsatellite stable (MSS) CRC. The BRAF V600E mutation has been associated with worse survival in MSS CRC. This mutation occurs in 40% of MSI CRC and it is unclear

Expression of angiogenic switch, cachexia and inflammation factors at the crossroad in undifferentiated thyroid carcinoma with BRAF(V600E).

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Husain, Amjad; Hu, Nina; Sadow, Peter M; Nucera, Carmelo

2016-10-01

Cachexia is the result of complex metabolic alterations which cause morbidity and mortality in patients with advanced cancers including undifferentiated (anaplastic) thyroid carcinoma (ATC). ATC is a lethal disease with limited therapeutic options and unclear etiology for cachexia. We hypothesize that the BRAF(V600E) oncoprotein triggers microvascular endothelial cell tubule formation (in vitro angiogenesis) by means of factors which play a crucial role in angiogenic switch, inflammation/immune response and cachexia. We use human ATC cells and applied multiplex ELISA assay to screen for and measure angiogenic/cachectic and pro-inflammatory factors in the ATC-derived secretome. We find that vemurafenib anti-BRAF(V600E) therapy significantly reduces secreted VEGFA, VEGFC and IL6 protein levels compared to vehicle-treated ATC cells. As a result, the secretome from vemurafenib-treated ATC cells inhibits microvascular endothelial cell-related in vitro angiogenesis. Furthermore, ATC clinical samples express VEGFA, VEGFC and IL6 proteins. Our results suggest that angiogenic/cachectic and pro-inflammatory/immune response factors could play a crucial role in BRAF(V600E)-positive human ATC aggressiveness. Understanding the extent to which microenvironment-associated angiogenic factors participate in cachexia and cancer metabolism in advanced thyroid cancers will reveal new biomarkers and foster novel therapeutic approaches. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A metastatic colon adenocarcinoma harboring BRAF V600E has a durable major response to dabrafenib/trametinib and chemotherapy

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Williams CB

2015-12-01

Full Text Available Casey B Williams,1,* Caitlin McMahon,2,* Siraj M Ali,2 Mark Abramovitz,1 Kirstin A Williams,1 Jessica Klein,1 Heidi McKean,1 Roman Yelensky,2 Thomas J George Jr,3 Julia A Elvin,2 Salil Soman,4 Doron Lipson,2 Juliann Chmielecki,2 Deborah Morosini,2 Vincent A Miller,2 Philip J Stephens,2 Jeffrey S Ross,2,5 Brian Leyland-Jones1 1Avera Cancer Institute, Sioux Falls, SD, USA; 2Foundation Medicine, Inc., Cambridge, MA, USA; 3University of Florida College of Medicine, Gainesville, FL, USA; 4Beth Israel Deaconess Medical Center, Boston, MA, USA; 5Albany Medical College, Albany, NY, USA *These authors contributed equally to this work Abstract: The subset of metastatic colorectal adenocarcinomas that harbor BRAF V600E mutations are aggressive tumors with significantly shortened survival and limited treatment options. Here we present a colorectal cancer patient whose disease progressed through standard chemotherapy and who developed liver metastasis. Comprehensive genomic profiling (FoundationOne® identified a BRAF V600E mutation in the liver lesion, as well as other genomic alterations consistent with colorectal cancers. Combination therapy of dabrafenib and trametinib with standard cytotoxic chemotherapy resulted in a durable major ongoing response for the patient. This report illustrates the utility of comprehensive genomic profiling with personalized targeted therapy for aggressive metastatic colorectal adenocarcinomas. Keywords: oxaliplatin, colorectal adenocarcinoma, combination targeted therapy, BRAF mutations

Landscape of Targeted Anti-Cancer Drug Synergies in Melanoma Identifies a Novel BRAF-VEGFR/PDGFR Combination Treatment.

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Adam A Friedman

Full Text Available A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1 transporter inhibition. We identified drugs sensitizing cell lines that are BRAFV600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR and platelet derived growth factor receptor (PDGFR family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis in vitro and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs, demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.

Single-Agent Panitumumab in Frail Elderly Patients With Advanced RAS and BRAF Wild-Type Colorectal Cancer: Challenging Drug Label to Light Up New Hope

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Cremolini, Chiara; Aprile, Giuseppe; Lonardi, Sara; Orlandi, Armando; Mennitto, Alessia; Berenato, Rosa; Antoniotti, Carlotta; Casagrande, Mariaelena; Marsico, Valentina; Marmorino, Federica; Cardellino, Giovanni Gerardo; Bergamo, Francesca; Tomasello, Gianluca; Formica, Vincenzo; Longarini, Raffaella; Giommoni, Elisa; Caporale, Marta; Di Bartolomeo, Maria; Loupakis, Fotios; de Braud, Filippo

2015-01-01

Background. No prospective trials have specifically addressed the efficacy and safety of panitumumab in elderly patients with metastatic colorectal cancer (CRC). We aimed at assessing the efficacy and safety of single agent panitumumab in “frail” elderly patients diagnosed with metastatic RAS and BRAF wild-type CRC. Materials and Methods. Forty elderly patients (aged ≥75 years) with metastatic RAS-BRAF wild-type CRC received off-label prescriptions of single-agent panitumumab at seven Italian institutions. Treatment was administered as first line in patients with absolute contraindication to any chemotherapy or as second-line treatment after failure of a fluoropyrimidine-based treatment, in the presence of contraindication to irinotecan. The outcome measures included objective response rate (ORR), as well as progression-free survival (PFS), disease control rate (DCR), overall survival (OS), and safety. Results. The median PFS and OS were 6.4 months (95% confidence interval [CI]: 4.9–8 months) and 14.3 months (95% CI: 10.9–17.7 months), respectively. ORR was 32.5%, and DCR was 72.5%. Dose reductions related to adverse events (AEs) were reported in 9 (23%) patients, but no permanent treatment discontinuation caused by was reported. The most frequent grade 3 AE was skin rash, with an incidence of 20%. Conclusion. Panitumumab is effective and well-tolerated in frail elderly patients with RAS-BRAF wild-type metastatic CRC and deemed unfit for chemotherapy. A randomized study is needed to confirm these data. Implications for Practice: Treatment of elderly patients with metastatic colorectal cancer represents a difficult challenge in clinical practice. A significant proportion of frail elderly patients do not receive treatment, reflecting ongoing uncertainty of clinical benefit and toxicity of chemotherapy. Unfit condition in this cohort of patients further limits antineoplastic prescription and consequently patient survival. RAS and BRAF wild-type status could

B vitamins, methionine and alcohol intake and risk of colon cancer in relation to BRAF mutation and CpG island methylator phenotype (CIMP).

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Schernhammer, Eva S; Giovannucci, Edward; Baba, Yoshifumi; Fuchs, Charles S; Ogino, Shuji

2011-01-01

One-carbon metabolism appears to play an important role in DNA methylation reaction. Evidence suggests that a low intake of B vitamins or high alcohol consumption increases colorectal cancer risk. How one-carbon nutrients affect the CpG island methylator phenotype (CIMP) or BRAF mutation status in colon cancer remains uncertain. Utilizing incident colon cancers in a large prospective cohort of women (the Nurses' Health Study), we determined BRAF status (N = 386) and CIMP status (N = 375) by 8 CIMP-specific markers [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and 8 other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT-1, MINT-31, p14, and WRN). We examined the relationship between intake of one-carbon nutrients and alcohol and colon cancer risk, by BRAF mutation or CIMP status. Higher folate intake was associated with a trend towards low risk of CIMP-low/0 tumors [total folate intake ≥400 µg/day vs. CIMP-high tumor risks (P(heterogeneity) = 0.73). Neither vitamin B(6), methionine or alcohol intake appeared to differentially influence risks for CIMP-high and CIMP-low/0 tumors. Using the 16-marker CIMP panel did not substantially alter our results. B vitamins, methionine or alcohol intake did not affect colon cancer risk differentially by BRAF status. This molecular pathological epidemiology study suggests that low level intake of folate may be associated with an increased risk of CIMP-low/0 colon tumors, but not that of CIMP-high tumors. However, the difference between CIMP-high and CIMP-low/0 cancer risks was not statistically significant, and additional studies are necessary to confirm these observations.

Somatic activating ARAF mutations in Langerhans cell histiocytosis

NARCIS (Netherlands)

Nelson, David S.; Quispel, Willemijn; Badalian-Very, Gayane; van Halteren, Astrid G. S.; van den Bos, Cor; Bovée, Judith V. M. G.; Tian, Sara Y.; van Hummelen, Paul; Ducar, Matthew; MacConaill, Laura E.; Egeler, R. Maarten; Rollins, Barrett J.

2014-01-01

The extracellular signal-regulated kinase (ERK) signaling pathway is activated in Langerhans cell histiocytosis (LCH) histiocytes, but only 60% of cases carry somatic activating mutations of BRAF. To identify other genetic causes of ERK pathway activation, we performed whole exome sequencing on

Rapid BRAF mutation tests in patients with advanced melanoma : Comparison of immunohistochemistry, Droplet Digital PCR, and the Idylla Mutation Platform

NARCIS (Netherlands)

Bisschop, Cornelis; Ter Elst, Arja; Bosman, Lisette J; Platteel, Inge; Jalving, Mathilde; van den Berg, Anke; Diepstra, Arjan; van Hemel, Bettien; Diercks, Gilles F H; Hospers, Geke A P; Schuuring, Ed

BRAF mutational testing has become a common practice in the diagnostic process of patients with advanced melanoma. Although time-consuming, DNA sequencing techniques are the current gold standard for mutational testing. However, in certain clinical situations, a rapid test result is required. In

BRAF V600E-Positive Multisite Langerhans Cell Histiocytosis in a Preterm Neonate

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Sara V. Bates

2013-10-01

Full Text Available Hemorrhagic pustules with a “blueberry muffin” appearance accompanied by respiratory failure in a neonate present a challenging differential diagnosis that includes infections and neoplasms. We present a case of multiorgan, multisite Langerhans cell histiocytosis (LCH, positive for the oncogenic BRAF V600E mutation, in a preterm neonate. Infants with LCH pose a diagnostic challenge due to their heterogeneous presentations. This case is unusual in that the newborn presented with severe multiorgan involvement. Due to the rare incidence, wide spectrum of clinical manifestations, and high mortality rate, clinicians must maintain a high index of suspicion for LCH.

CpG island methylator phenotype identifies high risk patients among microsatellite stable BRAF mutated colorectal cancers.

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Vedeld, Hege Marie; Merok, Marianne; Jeanmougin, Marine; Danielsen, Stine A; Honne, Hilde; Presthus, Gro Kummeneje; Svindland, Aud; Sjo, Ole H; Hektoen, Merete; Eknaes, Mette; Nesbakken, Arild; Lothe, Ragnhild A; Lind, Guro E

2017-09-01

The prognostic value of CpG island methylator phenotype (CIMP) in colorectal cancer remains unsettled. We aimed to assess the prognostic value of this phenotype analyzing a total of 1126 tumor samples obtained from two Norwegian consecutive colorectal cancer series. CIMP status was determined by analyzing the 5-markers CAGNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 by quantitative methylation specific PCR (qMSP). The effect of CIMP on time to recurrence (TTR) and overall survival (OS) were determined by uni- and multivariate analyses. Subgroup analyses were conducted according to MSI and BRAF mutation status, disease stage, and also age at time of diagnosis (CIMP positive tumors demonstrated significantly shorter TTR and worse OS compared to those with CIMP negative tumors (multivariate hazard ratio [95% CI] 1.86 [1.31-2.63] and 1.89 [1.34-2.65], respectively). In stratified analyses, CIMP tumors showed significantly worse outcome among patients with microsatellite stable (MSS, P CIMP is significantly associated with inferior outcome for colorectal cancer patients, and can stratify the poor prognostic patients with MSS BRAF mutated tumors. © 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Gastric Medullary Carcinoma with Sporadic Mismatch Repair Deficiency and a TP53 R273C Mutation: An Unusual Case with Wild-Type BRAF

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Brett M. Lowenthal

2017-01-01

Full Text Available Medullary carcinoma has long been recognized as a subtype of colorectal cancer associated with microsatellite instability and Lynch syndrome. Gastric medullary carcinoma is a very rare neoplasm. We report a 67-year-old male who presented with a solitary gastric mass. Total gastrectomy revealed a well-demarcated, poorly differentiated carcinoma with an organoid growth pattern, pushing borders, and abundant peritumoral lymphocytic response. The prior cytology was cellular with immunohistochemical panel consistent with upper gastrointestinal/pancreaticobiliary origin. Overall, the histopathologic findings were consistent with gastric medullary carcinoma. A mismatch repair panel revealed a mismatch repair protein deficient tumor with loss of MLH1 and PMS2 expression. BRAF V600E immunostain (VE1 and BRAF molecular testing were negative, indicating a wild-type gene. Tumor sequencing of MLH1 demonstrated a wild-type gene, while our molecular panel identified TP53 c.817C>T (p.R273C mutation. These findings were compatible with a sporadic tumor. Given that morphologically identical medullary tumors often occur in Lynch syndrome, it is possible that mismatch repair loss is an early event in sporadic tumors with p53 mutation being a late event. Despite having wild-type BRAF, this tumor is sporadic and unrelated to Lynch syndrome. This case report demonstrates that coordinate ancillary studies are needed to resolve sporadic versus hereditary rare tumors.

Health-related quality of life in patients with metastatic colorectal cancer, association with systemic inflammatory response and RAS and BRAF mutation status

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Thomsen, Maria; Guren, Marianne Grønlie; Skovlund, Eva

2017-01-01

Background The aim of this study was to evaluate the effect of cetuximab on health-related quality of life (HRQoL) in the NORDIC-VII trial on metastatic colorectal cancer (mCRC), and to assess HRQoL in relation to RAS and BRAF mutation status and inflammatory biomarkers. Patient and methods HRQo....... There was a statistically significant association between reduction in IL-6 and CRP levels and improvement in HRQoL during treatment from baseline to cycle 4. Conclusion The addition of cetuximab to chemotherapy did not affect HRQoL in mCRC patients. Patients with BRAF-mutated tumours have both a worse prognosis and a poor...

B vitamins, methionine and alcohol intake and risk of colon cancer in relation to BRAF mutation and CpG island methylator phenotype (CIMP.

Directory of Open Access Journals (Sweden)

Eva S Schernhammer

Full Text Available One-carbon metabolism appears to play an important role in DNA methylation reaction. Evidence suggests that a low intake of B vitamins or high alcohol consumption increases colorectal cancer risk. How one-carbon nutrients affect the CpG island methylator phenotype (CIMP or BRAF mutation status in colon cancer remains uncertain.Utilizing incident colon cancers in a large prospective cohort of women (the Nurses' Health Study, we determined BRAF status (N = 386 and CIMP status (N = 375 by 8 CIMP-specific markers [CACNA1G, CDKN2A (p16, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and 8 other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT-1, MINT-31, p14, and WRN. We examined the relationship between intake of one-carbon nutrients and alcohol and colon cancer risk, by BRAF mutation or CIMP status.Higher folate intake was associated with a trend towards low risk of CIMP-low/0 tumors [total folate intake ≥400 µg/day vs. <200 µg/day; the multivariate relative risk = 0.73; 95% CI = 0.53-1.02], whereas total folate intake had no influence on CIMP-high tumor risks (P(heterogeneity = 0.73. Neither vitamin B(6, methionine or alcohol intake appeared to differentially influence risks for CIMP-high and CIMP-low/0 tumors. Using the 16-marker CIMP panel did not substantially alter our results. B vitamins, methionine or alcohol intake did not affect colon cancer risk differentially by BRAF status.This molecular pathological epidemiology study suggests that low level intake of folate may be associated with an increased risk of CIMP-low/0 colon tumors, but not that of CIMP-high tumors. However, the difference between CIMP-high and CIMP-low/0 cancer risks was not statistically significant, and additional studies are necessary to confirm these observations.

The combination of MLN2238 (ixazomib) with interferon-alpha results in enhanced cell death in melanoma.

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Suarez-Kelly, Lorena P; Kemper, Gregory M; Duggan, Megan C; Stiff, Andrew; Noel, Tiffany C; Markowitz, Joseph; Luedke, Eric A; Yildiz, Vedat O; Yu, Lianbo; Jaime-Ramirez, Alena Cristina; Karpa, Volodymyr; Zhang, Xiaoli; Carson, William E

2016-12-06

The ubiquitin-proteasome signaling pathway is critical for cell cycle regulation and neoplastic growth. Proteasome inhibition can activate apoptotic pathways. Bortezomib, a selective proteasome inhibitor, has anti-melanoma activity. MLN2238 (ixazomib), an oral proteasome inhibitor, has improved pharmacotherapeutic parameters compared to bortezomib. Interferon-alpha (IFN-α), an immune boosting agent, is FDA-approved for treatment of melanoma. In this study in vitro and in vivo evaluation of the antitumor potential of ixazomib and combination treatments with ixazomib and IFN-α were performed. Apoptosis induced by ixazomib was first observed at 12 hours and was maximal at 48 hours with similar levels of cell death compared to bortezomib. IFN-α alone had little effect on cell viability in vitro. However, the combination of ixazomib with IFN-α significantly enhanced ixazomib's ability to induce apoptotic cell death in BRAF V600E mutant and BRAF wild-type human melanoma tumor cells. The combination of ixazomib and IFN-α also enhanced inhibition of cell proliferation in BRAF V600E mutant melanoma tumor cells; however, this was not seen in BRAF wild-type cells. Ixazomib-induced apoptosis was associated with processing of the pro-apoptotic proteins procaspase-3, -7, -8, and -9, and cleavage of poly-ADP-ribose polymerase (PARP). In an in vivo xenograft model of human melanoma, combination treatment with IFN-α-2b and ixazomib demonstrated a significant reduction in tumor volume when compared to vehicle (p = 0.005) and single therapy ixazomib (p = 0.017) and IFN-α-2b (p = 0.036). These pre-clinical results support further evaluation of combination treatment with ixazomib and IFN-α for the treatment of advanced BRAF V600E mutant melanoma.

BRAF Mutation Is Associated With a Specific Cell Type With Features Suggestive of Senescence in Ovarian Serous Borderline (Atypical Proliferative) Tumors

DEFF Research Database (Denmark)

Zeppernick, Felix; Ardighieri, Laura; Hannibal, Charlotte G

2014-01-01

features could be reproduced in cultured epithelial cells by ectopic expression of BRAF(V600E). Senescence was further established by markers such as SA-β-gal staining, expression of p16 and p21, and reduction in DNA synthesis. In conclusion, this study sheds light on the pathogenesis of this unique group...

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

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Heightman, Tom D; Berdini, Valerio; Braithwaite, Hannah; Buck, Ildiko M; Cassidy, Megan; Castro, Juan; Courtin, Aurélie; Day, James E H; East, Charlotte; Fazal, Lynsey; Graham, Brent; Griffiths-Jones, Charlotte M; Lyons, John F; Martins, Vanessa; Muench, Sandra; Munck, Joanne M; Norton, David; O'Reilly, Marc; Palmer, Nick; Pathuri, Puja; Reader, Michael; Rees, David C; Rich, Sharna J; Richardson, Caroline; Saini, Harpreet; Thompson, Neil T; Wallis, Nicola G; Walton, Hugh; Wilsher, Nicola E; Woolford, Alison J-A; Cooke, Michael; Cousin, David; Onions, Stuart; Shannon, Jonathan; Watts, John; Murray, Christopher W

2018-05-31

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Molecular spectrum of KRAS, NRAS, BRAF, PIK3CA, TP53, and APC somatic gene mutations in Arab patients with colorectal cancer: determination of frequency and distribution pattern

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Al-Shamsi, Humaid O.; Jones, Jeremy; Fahmawi, Yazan; Dahbour, Ibrahim; Tabash, Aziz; Abdel-Wahab, Reham; Abousamra, Ahmed O. S.; Shaw, Kenna R.; Xiao, Lianchun; Hassan, Manal M.; Kipp, Benjamin R.; Kopetz, Scott; Soliman, Amr S.; McWilliams, Robert R.; Wolff, Robert A.

2016-01-01

Background The frequency rates of mutations such as KRAS, NRAS, BRAF, and PIK3CA in colorectal cancer (CRC) differ among populations. The aim of this study was to assess mutation frequencies in the Arab population and determine their correlations with certain clinicopathological features. Methods Arab patients from the Arab Gulf region and a population of age- and sex-matched Western patients with CRC whose tumors were evaluated with next-generation sequencing (NGS) were identified and retrospectively reviewed. The mutation rates of KRAS, NRAS, BRAF, PIK3CA, TP53, and APC were recorded, along with clinicopathological features. Other somatic mutation and their rates were also identified. Fisher’s exact test was used to determine the association between mutation status and clinical features. Results A total of 198 cases were identified; 99 Arab patients and 99 Western patients. Fifty-two point seven percent of Arab patients had stage IV disease at initial presentation, 74.2% had left-sided tumors. Eighty-nine point two percent had tubular adenocarcinoma and 10.8% had mucinous adenocarcinoma. The prevalence rates of KRAS, NRAS, BRAF, PIK3CA, TP53, APC, SMAD, FBXW7 mutations in Arab population were 44.4%, 4%, 4%, 13.1%, 52.5%, 27.3%, 2% and 3% respectively. Compared to 48.4%, 4%, 4%, 12.1%, 47.5%, 24.2%, 11.1% and 0% respectively in matched Western population. Associations between these mutations and patient clinicopathological features were not statistically significant. Conclusions This is the first study to report comprehensive hotspot mutations using NGS in Arab patients with CRC. The frequency of KRAS, NRAS, BRAF, TP53, APC and PIK3CA mutations were similar to reported frequencies in Western population except SMAD4 that had a lower frequency and higher frequency of FBXW7 mutation. PMID:28078112

MITF Modulates Therapeutic Resistance through EGFR Signaling.

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Ji, Zhenyu; Erin Chen, Yiyin; Kumar, Raj; Taylor, Michael; Jenny Njauw, Ching-Ni; Miao, Benchun; Frederick, Dennie T; Wargo, Jennifer A; Flaherty, Keith T; Jönsson, Göran; Tsao, Hensin

2015-07-01

Response to targeted therapies varies significantly despite shared oncogenic mutations. Nowhere is this more apparent than in BRAF (V600E)-mutated melanomas where initial drug response can be striking and yet relapse is commonplace. Resistance to BRAF inhibitors have been attributed to the activation of various receptor tyrosine kinases (RTKs), although the underlying mechanisms have been largely uncharacterized. Here, we found that EGFR-induced vemurafenib resistance is ligand dependent. We employed whole-genome expression analysis and discovered that vemurafenib resistance correlated with the loss of microphthalmia-associated transcription factor (MITF), along with its melanocyte lineage program, and with the activation of EGFR signaling. An inverse relationship between MITF, vemurafenib resistance, and EGFR was then observed in patient samples of recurrent melanoma and was conserved across melanoma cell lines and patients' tumor specimens. Functional studies revealed that MITF depletion activated EGFR signaling and consequently recapitulated the resistance phenotype. In contrast, forced expression of MITF in melanoma and colon cancer cells inhibited EGFR and conferred sensitivity to BRAF/MEK inhibitors. These findings indicate that an "autocrine drug resistance loop" is suppressed by melanocyte lineage signal(s), such as MITF. This resistance loop modulates drug response and could explain the unique sensitivity of melanomas to BRAF inhibition.

Associations between primary tumor RAS, BRAF and PIK3CA mutation status and metastatic site in patients with chemo-resistant metastatic colorectal cancer

DEFF Research Database (Denmark)

Christensen, Troels Dreier; Palshof, Jesper Andreas; Larsen, Finn Ole

2018-01-01

investigated the association between RAS (KRAS or NRAS), BRAF, PIK3CA mutations and metastatic pattern in patients with metastatic (m) CRC. MATERIAL AND METHODS: This study reviewed Danish biobank and database of patients with mCRC who received cetuximab and irinotecan, independent of RAS mutation status...

Differences in global DNA methylation of testicular seminoma are not associated with changes in histone modifications, clinical prognosis, BRAF mutations or gene expression

DEFF Research Database (Denmark)

Pedersen, Louise Holm; Nielsen, John E; Daugaard, Gedske

2016-01-01

-seminomas. It is well established from e.g. melanoma, colorectal and thyroid cancer that a methylated phenotype can be correlated to prognosis and can be related to BRAF mutations. In the present study we investigated the global methylation level in 67 seminomas and classified them as hypo-methylated, intermediate...

The role of hypoxia inducible factor-1 alpha in bypassing oncogene-induced senescence.

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Mehtap Kilic Eren

Full Text Available Oncogene induced senescence (OIS is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR, senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on RasV12-induced senescence in human diploid fibroblasts (HDFs. We showed here that hypoxia prevents execution of oncogene induced senescence (OIS, through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21CIP1 and p16INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α. In addition, hypoxia also decreased marks of H-RasV12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21CIP1 but not p16INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.

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Konermann, Silvana; Brigham, Mark D; Trevino, Alexandro E; Joung, Julia; Abudayyeh, Omar O; Barcena, Clea; Hsu, Patrick D; Habib, Naomi; Gootenberg, Jonathan S; Nishimasu, Hiroshi; Nureki, Osamu; Zhang, Feng

2015-01-29

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model.

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Joshi, Sandeep S; Jiang, Shunlin; Unni, Emmanual; Goding, Stephen R; Fan, Tao; Antony, Paul A; Hornyak, Thomas J

2018-01-01

Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of

17-AAG inhibits vemurafenib-associated MAP kinase activation and is synergistic with cellular immunotherapy in a murine melanoma model

Science.gov (United States)

Unni, Emmanual; Goding, Stephen R.; Fan, Tao; Antony, Paul A.; Hornyak, Thomas J.

2018-01-01

Heat shock protein 90 (HSP90) is a molecular chaperone which stabilizes client proteins with important roles in tumor growth. 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90 ATPase activity, occupies the ATP binding site of HSP90 causing a conformational change which destabilizes client proteins and directs them towards proteosomal degradation. Malignant melanomas have active RAF-MEK-ERK signaling which can occur either through an activating mutation in BRAF (BRAFV600E) or through activation of signal transduction upstream of BRAF. Prior work showed that 17-AAG inhibits cell growth in BRAFV600E and BRAF wildtype (BRAFWT) melanomas, although there were conflicting reports about the dependence of BRAFV600E and BRAFWT upon HSP90 activity for stability. Here, we demonstrate that BRAFWT and CRAF are bound by HSP90 in BRAFWT, NRAS mutant melanoma cells. HSP90 inhibition by 17-AAG inhibits ERK signaling and cell growth by destabilizing CRAF but not BRAFWT in the majority of NRAS mutant melanoma cells. The highly-selective BRAFV600E inhibitor, PLX4032 (vemurafenib), inhibits ERK signaling and cell growth in mutant BRAF melanoma cells, but paradoxically enhances signaling in cells with wild-type BRAF. In our study, we examined whether 17-AAG could inhibit PLX4032-enhanced ERK signaling in BRAFWT melanoma cells. As expected, PLX4032 alone enhanced ERK signaling in the BRAFWT melanoma cell lines Mel-Juso, SK-Mel-2, and SK-Mel-30, and inhibited signaling and cell growth in BRAFV600E A375 cells. However, HSP90 inhibition by 17-AAG inhibited PLX4032-enhanced ERK signaling and inhibited cell growth by destabilizing CRAF. Surprisingly, 17-AAG also stimulated melanin production in SK-Mel-30 cells and enhanced TYRP1 and DCT expression without stimulating TYR production in all three BRAFWT cell lines studied as well as in B16F10 mouse melanoma cells. In vivo, the combination of 17-AAG and cellular immunotherapy directed against Tyrp1 enhanced the inhibition of

PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling.

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Shrestha, Y; Schafer, E J; Boehm, J S; Thomas, S R; He, F; Du, J; Wang, S; Barretina, J; Weir, B A; Zhao, J J; Polyak, K; Golub, T R; Beroukhim, R; Hahn, W C

2012-07-19

Activating mutations in the RAS family or BRAF frequently occur in many types of human cancers but are rarely detected in breast tumors. However, activation of the RAS-RAF-MEK-ERK MAPK pathway is commonly observed in human breast cancers, suggesting that other genetic alterations lead to activation of this signaling pathway. To identify breast cancer oncogenes that activate the MAPK pathway, we screened a library of human kinases for their ability to induce anchorage-independent growth in a derivative of immortalized human mammary epithelial cells (HMLE). We identified p21-activated kinase 1 (PAK1) as a kinase that permitted HMLE cells to form anchorage-independent colonies. PAK1 is amplified in several human cancer types, including 30--33% of breast tumor samples and cancer cell lines. The kinase activity of PAK1 is necessary for PAK1-induced transformation. Moreover, we show that PAK1 simultaneously activates MAPK and MET signaling; the latter via inhibition of merlin. Disruption of these activities inhibits PAK1-driven anchorage-independent growth. These observations establish PAK1 amplification as an alternative mechanism for MAPK activation in human breast cancer and credential PAK1 as a breast cancer oncogene that coordinately regulates multiple signaling pathways, the cooperation of which leads to malignant transformation.

Illustrative cases for monitoring by quantitative analysis of BRAF/NRAS ctDNA mutations in liquid biopsies of metastatic melanoma patients who gained clinical benefits from anti-PD1 antibody therapy.

Science.gov (United States)

Seremet, Teofila; Planken, Simon; Schreuer, Max; Jansen, Yanina; Delaunoy, Mélanie; El Housni, Hakim; Lienard, Danielle; Del Marmol, Véronique; Heimann, Pierre; Neyns, Bart

2018-02-01

Anti-programmed death 1 (PD-1) monoclonal antibodies improve the survival of metastatic melanoma patients. Predictive or monitoring biomarkers for response to this therapy could improve the clinical management of these patients. To date, no established biomarkers are available for monitoring the response to immunotherapy. Tumor- specific mutations in circulating tumor DNA (ctDNA) such as BRAF and NRAS mutations for melanoma patients have been proposed for monitoring of immunotherapy response. We present seven illustrative cases for the use of ctDNA BRAF and NRAS mutations' monitoring in plasma. The cases described exemplify four distinct clinical benefit patterns: rapid and durable complete response (CR), early progression, followed by CR, CR followed by early progression after interrupting treatment and long-term disease stabilization. These representative cases suggest that comprehensive BRAF/NRAS ctDNA monitoring during anti-PD1 therapy is informative and can be of added value for the monitoring of melanoma patients gaining clinical benefit on anti-PD1 treatment. An important advantage of our approach is that using the cartridge system on the Idylla platform for mutation analysis, the results become available the same day 2 h after plasma collection. Therefore, in the future, the ctDNA level can be an element in the clinical management of the patients.

Analysis of BRAF V600E mutation in 1,320 nervous system tumors reveals high mutation frequencies in pleomorphic xanthoastrocytoma, ganglioglioma and extra-cerebellar pilocytic astrocytoma

NARCIS (Netherlands)

Schindler, G.; Capper, D.; Meyer, J.; Janzarik, W.; Omran, H.; Herold-Mende, C.; Schmieder, K.; Wesseling, P.; Mawrin, C.; Hasselblatt, M.; Louis, D.N.; Korshunov, A.; Pfister, S.; Hartmann, C.; Paulus, W.; Reifenberger, G.; Deimling, A. Von

2011-01-01

Missense mutations of the V600E type constitute the vast majority of tumor-associated somatic alterations in the v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) gene. Initially described in melanoma, colon and papillary thyroid carcinoma, these alterations have also been observed in primary

Disruption of IGF-1R signaling increases TRAIL-induced apoptosis: A new potential therapy for the treatment of melanoma

Energy Technology Data Exchange (ETDEWEB)

Karasic, Thomas B.; Hei, Tom K. [Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Ivanov, Vladimir N., E-mail: vni3@columbia.edu [Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States)

2010-07-15

Resistance of cancer cells to apoptosis is dependent on a balance of multiple genetic and epigenetic mechanisms, which up-regulate efficacy of the surviving growth factor-receptor signaling pathways and suppress death-receptor signaling pathways. The Insulin-like Growth Factor-1 Receptor (IGF-1R) signaling pathway is highly active in metastatic melanoma cells by mediating downstream activation of PI3K-AKT and MAPK pathways and controlling general cell survival and proliferation. In the present study, we used human melanoma lines with established genotypes that represented different phases of cancer development: radial-growth-phase WM35, vertical-growth-phase WM793, metastatic LU1205 and WM9 [1]. All these lines have normal NRAS. WM35, WM793, LU1205 and WM9 cells have mutated BRAF (V600E). WM35 and WM9 cells express normal PTEN, while in WM793 cells PTEN expression is down-regulated; finally, in LU1205 cells PTEN is inactivated by mutation. Cyclolignan picropodophyllin (PPP), a specific inhibitor of IGF-1R kinase activity, strongly down-regulated the basal levels of AKT activity in WM9 and in WM793 cells, modestly does so in LU1205, but has no effect on AKT activity in the early stage WM35 cells that are deficient in IGF-1R. In addition, PPP partially down-regulated the basal levels of active ERK1/2 in all lines used, highlighting the role of an alternative, non-BRAF pathway in MAPK activation. The final result of PPP treatment was an induction of apoptosis in WM793, WM9 and LU1205 melanoma cells. On the other hand, dose-dependent inhibition of IGF-1R kinase activity by PPP at a relatively narrow dose range (near 500 nM) has different effects on melanoma cells versus normal cells, inducing apoptosis in cancer cells and G2/M arrest of fibroblasts. To further enhance the pro-apoptotic effects of PPP on melanoma cells, we used a combined treatment of TNF-Related Apoptosis-Inducing Ligand (TRAIL) and PPP. This combination substantially increased death by apoptosis for

Baseline β-catenin, programmed death-ligand 1 expression and tumour-infiltrating lymphocytes predict response and poor prognosis in BRAF inhibitor-treated melanoma patients.

Science.gov (United States)

Massi, Daniela; Romano, Emanuela; Rulli, Eliana; Merelli, Barbara; Nassini, Romina; De Logu, Francesco; Bieche, Ivan; Baroni, Gianna; Cattaneo, Laura; Xue, Gongda; Mandalà, Mario

2017-06-01

The activation of oncogenic Wnt/β-catenin pathway in melanoma contributes to a lack of T-cell infiltration. Whether baseline β-catenin expression in the context of tumour-infiltrating lymphocytes (TILs) and programmed death ligand-1 (PD-L1) overexpression correlates with prognosis of metastatic melanoma patients (MMPs) treated with mitogen-activated protein kinase, MAPK inhibitor (MAPKi) monotherapy, however, has not been fully clarified. Sixty-four pre-treatment formalin-fixed and paraffin embedded melanoma samples from MMP treated with a BRAF inhibitor (n = 39) or BRAF and MEK inhibitors (n = 25) were assessed for presence of β-catenin, PD-L1, cluster of differentiation (CD)8, CD103 and forkhead box protein P3 (FOXP3) expression by immunohistochemistry, and results were correlated with clinical outcome. Quantitative assessment of mRNA transcripts associated with Wnt/β-catenin pathway and immune response was performed in 51 patients. We found an inverse correlation between tumoural β-catenin expression and the level of CD8, CD103 or forkhead box protein P3 (FOXP3) positivity in the tumour microenvironment (TME). By multivariate analysis, PD-L1 <5% (odds ratio, OR 0.12, 95% confidence interval, CI 0.03-0.53, p = 0.005) and the presence of CD8+ T cells (OR 18.27, 95%CI 2.54-131.52, p = 0.004) were significantly associated with a higher probability of response to MAPKi monotherapy. Responding patients showed a significantly increased expression of mRNA transcripts associated with adaptive immunity and antigen presentation. By multivariate analysis, progression-free survival (PFS) (hazards ratio (HR) = 0.25 95%CI 0.10-0.61, p = 0.002) and overall survival (OS) (HR = 0.24 95%CI 0.09-0.67, p = 0.006) were longer in patients with high density of CD8+ T cells and β-catenin <10% than those without CD8+ T cells infiltration and β-catenin ≥10%. Our findings provide evidence that in the context of MAPKi monotherapy, immune subsets in the (TME) and

LMW-E/CDK2 Deregulates Acinar Morphogenesis, Induces Tumorigenesis, and Associates with the Activated b-Raf-ERK1/2-mTOR Pathway in Breast Cancer Patients

Science.gov (United States)

Duong, MyLinh T.; Akli, Said; Wei, Caimiao; Wingate, Hannah F.; Liu, Wenbin; Lu, Yiling; Yi, Min; Mills, Gordon B.; Hunt, Kelly K.; Keyomarsi, Khandan

2012-01-01

Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2–associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E–expressing cells by inducing G1/S cell cycle arrest. LMW

Inhibition of ERK1/2 or AKT Activity Equally Enhances Radiation Sensitization in B16F10 Cells

Science.gov (United States)

Kalal, Bhuvanesh Sukhlal; Fathima, Faraz; Pai, Vinitha Ramanath; Sanjeev, Ganesh; Krishna, Chilakapati Murali; Upadhya, Dinesh

2018-01-01

Background The aim of the study was to evaluate the radiation sensitizing ability of ERK1/2, PI3K-AKT and JNK inhibitors in highly radiation resistant and metastatic B16F10 cells which carry wild-type Ras and Braf. Methods Mouse melanoma cell line B16F10 was exposed to 1.0, 2.0 and 3.0 Gy of electron beam radiation. Phosphorylated ERK1/2, AKT and JNK levels were estimated by ELISA. Cells were exposed to 2.0 and 3.0 Gy of radiation with or without prior pharmacological inhibition of ERK1/2, AKT as well as JNK pathways. Cell death induced by radiation as well as upon inhibition of these pathways was measured by TUNEL assay using flow cytometry. Results Exposure of B16F10 cells to 1.0, 2.0 and 3.0 Gy of electron beam irradiation triggered an increase in all the three phosphorylated proteins compared to sham-treated and control groups. B16F10 cells pre-treated with either ERK1/2 or AKT inhibitors equally enhanced radiation-induced cell death at 2.0 as well as 3.0 Gy (P < 0.001), while inhibition of JNK pathway increased radiation-induced cell death to a lesser extent. Interestingly combined inhibition of ERK1/2 or AKT pathways did not show additional cell death compared to individual ERK1/2 or AKT inhibition. This indicates that ERK1/2 or AKT mediates radiation resistance through common downstream molecules in B16F10 cells. Conclusions Even without activating mutations in Ras or Braf genes, ERK1/2 and AKT play a critical role in B16F10 cell survival upon radiation exposure and possibly act through common downstream effector/s. PMID:29581812

[Effect of ERK1/2 Signaling Pathway Inhibitor PD98059 on the Expression of Ras, BRaf, MEK, ERK1/2 in Marrow Nucleated Red Blood Cells of CMS Patients].

Science.gov (United States)

Han, Yuan-Fang; Ji, Lin-Hua; Feng, Ting-Ting; Liu, Fang; Cui, Sen; Su, Juan

2017-10-01

To investigate the effect of ERK1 / 2 signaling pathway inhibitor PD98059 on Ras, Raf, MEK, ERK1, ERK2 expression in order to explore a new way for basic research and clinical treatment of the chronic mountain sickness(CMS). Sixteen CMS patients were selected, the bone marrow was collected for isolation of monomuclear cells (MNC), the cells were sorted by using CD71 and CD235a antibody magnetic beads, then positive cells were diveded into 5 groups: blank control, DMSO and PD98059 5, 10 and 20 µmol/L, and were cultured in hypoxid condition for 72 hours. The Ras-GTP levels in supernatant was detected by ELISA, the RT-PCR was used to determine the expression of BRaf, MEK, ERK1, ERK2 mRNA in nucleated red blood cells, and the Western blot method was used to detect expression of BRaf, MEK, ERK1, ERK2 protein. PD98059 had no effect on the level of Ras-GTP in each groups. Compared with the blank control group, the expression levels of BRaf, MEK mRNA in DMSO group were not statistically significant (P values were 0.826, 0.298). Compared with the PD98059 20 mol/L group, the expression level of ERK1/2 mRNA was statistically significant (P=0.001, 0.002). Compared with the blank control group, expression levels of p-BRaf, p-MEK protein in DMSO group were not statistically significant (P=0.370, 0.351). Compared with the PD98059 20 mol/L group, the difference of p-ERK1/2 protein level in other 4 groups were statistically significant (P values were <0.001, 0.007). PD98059 can up-regulate the expressions of ERK1/2 miRNA and p-ERK1/2 protein in bone marrow nucleated red blood cells, the Ras / Raf / MEK / ERK 1/2 pathway is the main signal transduction pathway in regulating bone marrow nucleated red blood cells, suggesting that Ras/Raf /MEK /ERK 1/2 pathway may be involved in the pathogenesis of chronic mountain sickness process.

BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

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Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F; Niu, Huifeng

2012-01-01

Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.

BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

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Brenden Chen

Full Text Available Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167 or mTORC1 inhibitor (rapamycin induced AKT phosphorylation (pAKT and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2 and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.

Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

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Tongfang Tang

Full Text Available BACKGROUND: Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD through alternation of liver innate immune response. AIMS: The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. METHODS: Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. RESULTS: High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4 expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. CONCLUSION: High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

Pro-inflammatory activated Kupffer cells by lipids induce hepatic NKT cells deficiency through activation-induced cell death.

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Tang, Tongfang; Sui, Yongheng; Lian, Min; Li, Zhiping; Hua, Jing

2013-01-01

Dietary lipids play an important role in the progression of non-alcoholic fatty liver disease (NAFLD) through alternation of liver innate immune response. The present study was to investigate the effect of lipid on Kupffer cells phenotype and function in vivo and in vitro. And further to investigate the impact of lipid on ability of Kupffer cell lipid antigen presentation to activate NKT cells. Wild type male C57BL/6 mice were fed either normal or high-fat diet. Hepatic steatosis, Kupffer cell abundance, NKT cell number and cytokine gene expression were evaluated. Antigen presentation assay was performed with Kupffer cells treated with certain fatty acids in vitro and co-cultured with NKT cells. High-fat diet induced hepatosteatosis, significantly increased Kupffer cells and decreased hepatic NKT cells. Lipid treatment in vivo or in vitro induced increase of pro-inflammatory cytokines gene expression and toll-like receptor 4 (TLR4) expression in Kupffer cells. Kupffer cells expressed high levels of CD1d on cell surface and only presented exogenous lipid antigen to activate NKT cells. Ability of Kupffer cells to present antigen and activate NKT cells was enhanced after lipid treatment. In addition, pro-inflammatory activated Kupffer cells by lipid treatment induced hepatic NKT cells activation-induced apoptosis and necrosis. High-fat diet increase Kupffer cells number and induce their pro-inflammatory status. Pro-inflammatory activated Kupfffer cells by lipid promote hepatic NKT cell over-activation and cell death, which lead to further hepatic NKT cell deficiency in the development of NAFLD.

Amelanotic melanoma presenting with plasmacytoid morphology and BRAF V600 mutation

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Linda Kocovski

2015-06-01

Full Text Available Plasmacytoid melanoma is an unusual variant of malignant melanoma. The plasmacytoid morphology can be found in a variety of other malignancies including carcinomas, plasma cell neoplasms, lymphoproliferative disorders, and sarcomas. The authors report a rare case of plasmacytoid amelanotic malignant melanoma in a 78-year-old man presenting with an enlarging palpable, erythematous mass on his left posterior shoulder. A fine needle aspirate showed atypical findings with single amelanotic cells with high nuclear to cytoplasmic ratio, mono- and multi-nucleation with prominent nucleoli and intranuclear inclusions. Review of the excision and immunohistochemical analysis revealed the malignant plasmacytoid cells stained with vimentin, S-100, HMB-45, and other staining patterns consistent with melanoma. Initial evaluation was negative for other sites of disease. However, 4 months later, the patient was noted to have metastatic disease to his lungs and liver. Given that the tumor was noted to be BRAF V600R mutated, the patient was started on single agent dabrafenib. The plasmacytoid morphology can be found in a variety of malignancies. Melanoma should be considered in the differential diagnosis of any malignancy presenting with plasmacytoid features.

Modification, biological evaluation and 3D QSAR studies of novel 2-(1,3-diaryl- 4,5-dihydro-1H-pyrazol-5-ylphenol derivatives as inhibitors of B-Raf kinase.

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Yu-Shun Yang

Full Text Available A series of novel 2-(1,3-diaryl- 4,5-dihydro-1H-pyrazol-5-ylphenol derivatives (C1-C24 have been synthesized. The B-Raf inhibitory activity and anti-proliferation activity of these compounds have been tested. Compound C6 displayed the most potent biological activity against B-RafV600E (IC50 = 0.15 µM and WM266.4 human melanoma cell line (GI50 = 1.75 µM, being comparable with the positive control (Vemurafenib and Erlotinib and more potent than our previous best compounds. The docking simulation was performed to analyze the probable binding models and poses while the QSAR model was built to check the previous work as well as to introduce new directions. This work aimed at seeking more potent inhibitors as well as discussing some previous findings. As a result, the introduction of ortho-hydroxyl group on 4,5-dihydro-1H-pyrazole skeleton did reinforce the anti-tumor activity while enlarging the group on N-1 of pyrazoline was also helpful.

Distribution of induced activity in tungsten targets

International Nuclear Information System (INIS)

Donahue, R.J.; Nelson, W.R.

1988-09-01

Estimates are made of the induced activity created during high-energy electron showers in tungsten, using the EGS4 code. Photon track lengths, neutron yields and spatial profiles of the induced activity are presented. 8 refs., 9 figs., 1 tab

A HRM assay for identification of low level BRAF V600E and V600K mutations using the CADMA principle in FFPE specimens.

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Huebner, Claudia; Weber, Remeny; Lloydd, Richard

2017-12-01

Melanoma patients with BRAF V600E and V600K mutations show complete or partial response to vemurafenib. Detection assays often scan for the common V600E mutation rather than the rare V600K variant, although this mutation can be found in a high proportion of melanoma patients in the South Pacific. Herein, we describe a BRAF high resolution melting (HRM) assay that can differentiate low level of V600E and V600K mutations using formalin fixed, paraffin embedded (FFPE) reference standards for assay validation. The assay is based on the competitive amplification of differentially melting amplicons (CADMA principle) and has a limit of detection of 0.8% mutant allele for V600K and 1.4% mutant allele for V600E. A differentiation between the two mutations based on the melting profile is possible even at low mutation level. Sixty FFPE specimens were scanned and mutations could be scored correctly as confirmed by castPCR. In summary, the developed HRM assay is suitable for detection of V600K and V600E mutations and proved to be reliable and cost effective in a diagnostic environment. Copyright © 2017 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.

Avoiding Severe Toxicity From Combined BRAF Inhibitor and Radiation Treatment: Consensus Guidelines from the Eastern Cooperative Oncology Group (ECOG)

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Anker, Christopher J., E-mail: chris.anker@UVMHealth.org [Division of Radiation Oncology, University of Vermont Cancer Center, Burlington, Vermont (United States); Grossmann, Kenneth F. [Division of Medical Oncology, Department of Medicine, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah (United States); Atkins, Michael B. [Department of Oncology, Georgetown-Lombardi Comprehensive Cancer Center, Georgetown University School of Medicine, Washington, District of Columbia (United States); Suneja, Gita [Department of Radiation Oncology, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah (United States); Tarhini, Ahmad A.; Kirkwood, John M. [Division of Hematology-Oncology, Department of Medicine, University of Pittsburgh Cancer Institute, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania (United States)

2016-06-01

BRAF kinase gene V600 point mutations drive approximately 40% to 50% of all melanomas, and BRAF inhibitors (BRAFi) have been found to significantly improve survival outcomes. Although radiation therapy (RT) provides effective symptom palliation, there is a lack of toxicity and efficacy data when RT is combined with BRAFi, including vemurafenib and dabrafenib. This literature review provides a detailed analysis of potential increased dermatologic, pulmonary, neurologic, hepatic, esophageal, and bowel toxicity from the combination of BRAFi and RT for melanoma patients described in 27 publications. Despite 7 publications noting potential intracranial neurotoxicity, the rates of radionecrosis and hemorrhage from whole brain RT (WBRT), stereotactic radiosurgery (SRS), or both do not appear increased with concurrent or sequential administration of BRAFis. Almost all grade 3 dermatitis reactions occurred when RT and BRAFi were administered concurrently. Painful, disfiguring nondermatitis cutaneous reactions have been described from concurrent or sequential RT and BRAFi administration, which improved with topical steroids and time. Visceral toxicity has been reported with RT and BRAFi, with deaths possibly related to bowel perforation and liver hemorrhage. Increased severity of radiation pneumonitis with BRAFi is rare, but more concerning was a potentially related fatal pulmonary hemorrhage. Conversely, encouraging reports have described patients with leptomeningeal spread and unresectable lymphadenopathy rendered disease free from combined RT and BRAFi. Based on our review, the authors recommend holding RT ≥3 days before and after fractionated RT and ≥1 day before and after SRS. No fatal reactions have been described with a dose <4 Gy per fraction, and time off systemic treatment should be minimized. Future prospective data will serve to refine these recommendations.

Enhanced stimulus-induced gamma activity in humans during propofol-induced sedation.

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Neeraj Saxena

Full Text Available Stimulus-induced gamma oscillations in the 30-80 Hz range have been implicated in a wide number of functions including visual processing, memory and attention. While occipital gamma-band oscillations can be pharmacologically modified in animal preparations, pharmacological modulation of stimulus-induced visual gamma oscillations has yet to be demonstrated in non-invasive human recordings. Here, in fifteen healthy humans volunteers, we probed the effects of the GABAA agonist and sedative propofol on stimulus-related gamma activity recorded with magnetoencephalography, using a simple visual grating stimulus designed to elicit gamma oscillations in the primary visual cortex. During propofol sedation as compared to the normal awake state, a significant 60% increase in stimulus-induced gamma amplitude was seen together with a 94% enhancement of stimulus-induced alpha suppression and a simultaneous reduction in the amplitude of the pattern-onset evoked response. These data demonstrate, that propofol-induced sedation is accompanied by increased stimulus-induced gamma activity providing a potential window into mechanisms of gamma-oscillation generation in humans.

Activating PTEN by COX-2 inhibitors antagonizes radiation-induced AKT activation contributing to radiosensitization

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Meng, Zhen [Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China); Department of Oral & Maxillofacial Surgery, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China); Gan, Ye-Hua, E-mail: kqyehuagan@bjmu.edu.cn [Central Laboratory, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China); Department of Oral & Maxillofacial Surgery, Peking University School and Hospital of Stomatology, 22 Zhongguancun Avenue South, Haidian District, Beijing 100081 (China)

2015-05-01

Radiotherapy is still one of the most effective nonsurgical treatments for many tumors. However, radioresistance remains a major impediment to radiotherapy. Although COX-2 inhibitors can induce radiosensitization, the underlying mechanism is not fully understood. In this study, we showed that COX-2 selective inhibitor celecoxib enhanced the radiation-induced inhibition of cell proliferation and apoptosis in HeLa and SACC-83 cells. Treatment with celecoxib alone dephosphorylated phosphatase and tensin homolog deleted on chromosome ten (PTEN), promoted PTEN membrane translocation or activation, and correspondingly dephosphorylated or inactivated protein kinase B (AKT). By contrast, treatment with radiation alone increased PTEN phosphorylation, inhibited PTEN membrane translocation and correspondingly activated AKT in the two cell lines. However, treatment with celecoxib or another COX-2 selective inhibitor (valdecoxib) completely blocked radiation-induced increase of PTEN phosphorylation, rescued radiation-induced decrease in PTEN membrane translocation, and correspondingly inactivated AKT. Moreover, celecoxib could also upregulate PTEN protein expression by downregulating Sp1 expression, thereby leading to the activation of PTEN transcription. Our results suggested that COX-2 inhibitors could enhance radiosensitization at least partially by activating PTEN to antagonize radiation-induced AKT activation. - Highlights: • COX-2 inhibitor, celecoxib, could enhance radiosensitization. • Radiation induced PTEN inactivation (phosphorylation) and AKT activation. • COX-2 inhibitor induced PTEN expression and activation, and inactivated AKT. • COX-2 inhibitor enhanced radiosensitization through activating PTEN.

Identification of novel 3,5-diarylpyrazoline derivatives containing salicylamide moiety as potential anti-melanoma agents.

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Li, Qing-Shan; Lv, Xian-Hai; Zhang, Yan-Bin; Dong, Jing-Jun; Zhou, Wen-Ping; Yang, Yang; Zhu, Hai-Liang

2012-11-01

There is an accumulating body of experimental evidences validating oncogenic BRAF(V600E) as a therapeutic target and offering opportunities for anti-melanoma drug development. Encouraged by the positive results of pyrazole derivatives as BRAF(V600E) inhibitors, we sought to design diverse novel potential BRAF(V600E) inhibitors as antitumor agents based on pyrazole skeleton. In silico and in vitro screening of our designed pyrazole derivatives has identified Hit 1 as BRAF(V600E) inhibitor. Based on its structure and through further structure modification, compound 25, which exhibited the most potent inhibitory activity with an IC(50) value of 0.16 μM for BRAF(V600E) and GI(50) value of 0.24 μM for mutant BRAF-dependent melanoma cells, was obtained. The 3D-QSAR models and the molecular docking simulation were introduced to analyze the structure-activity relationship. Copyright © 2012 Elsevier Ltd. All rights reserved.

Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death

International Nuclear Information System (INIS)

Guo, Shiguang; Mao, Li; Ji, Feng; Wang, Shouguo; Xie, Yue; Fei, Haodong; Wang, Xiao-dong

2016-01-01

Excessive glucocorticoid (GC) usage may lead to non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) exerts cytotoxic effect to cultured osteoblasts. Here, we investigated the potential activity of Compound 13 (C13), a novel α1 selective AMP-activated protein kinase (AMPK) activator, against the process. Our data revealed that C13 pretreatment significantly attenuated Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. AMPK activation mediated C13′ cytoprotective effect in osteoblasts. The AMPK inhibitor Compound C, shRNA-mediated knockdown of AMPKα1, or dominant negative mutation of AMPKα1 (T172A) almost abolished C13-induced AMPK activation and its pro-survival effect in osteoblasts. On the other hand, forced AMPK activation by adding AMPK activator A-769662 or exogenous expression a constitutively-active (ca) AMPKα1 (T172D) mimicked C13's actions and inhibited Dex-induced osteoblast cell death. Meanwhile, A-769662 or ca-AMPKα1 almost nullified C13's activity in osteoblast. Further studies showed that C13 activated AMPK-dependent nicotinamide adenine dinucleotide phosphate (NADPH) pathway to inhibit Dex-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary murine osteoblasts. Such effects by C13 were almost reversed by Compound C or AMPKα1 depletion/mutation. Together, these results suggest that C13 alleviates Dex-induced osteoblast cell death via activating AMPK signaling pathway. - Highlights: • Compound 13 (C13) attenuates dexamethasone (Dex)-induced osteoblast cell death. • C13-induced cytoprotective effect against Dex in osteoblasts requires AMPK activation. • Forced AMPK activation protects osteoblasts from Dex, nullifying C13's activities. • C13 increases NADPH activity and inhibits Dex-induced oxidative stress in osteoblasts.

Activating AMP-activated protein kinase by an α1 selective activator compound 13 attenuates dexamethasone-induced osteoblast cell death

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Guo, Shiguang [Department of Intensive Care Unit, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Mao, Li [Department of Endocrinology, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Ji, Feng, E-mail: huaiaifengjidr@163.com [Department of Orthopedics, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Wang, Shouguo; Xie, Yue; Fei, Haodong [Department of Orthopedics, Huai' an First People' s Hospital, Nanjing Medical University, Huai' an (China); Wang, Xiao-dong, E-mail: xiaodongwangsz@163.com [The Center of Diagnosis and Treatment for Children' s Bone Diseases, The Children' s Hospital Affiliated to Soochow University, Suzhou (China)

2016-03-18

Excessive glucocorticoid (GC) usage may lead to non-traumatic femoral head osteonecrosis. Dexamethasone (Dex) exerts cytotoxic effect to cultured osteoblasts. Here, we investigated the potential activity of Compound 13 (C13), a novel α1 selective AMP-activated protein kinase (AMPK) activator, against the process. Our data revealed that C13 pretreatment significantly attenuated Dex-induced apoptosis and necrosis in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. AMPK activation mediated C13′ cytoprotective effect in osteoblasts. The AMPK inhibitor Compound C, shRNA-mediated knockdown of AMPKα1, or dominant negative mutation of AMPKα1 (T172A) almost abolished C13-induced AMPK activation and its pro-survival effect in osteoblasts. On the other hand, forced AMPK activation by adding AMPK activator A-769662 or exogenous expression a constitutively-active (ca) AMPKα1 (T172D) mimicked C13's actions and inhibited Dex-induced osteoblast cell death. Meanwhile, A-769662 or ca-AMPKα1 almost nullified C13's activity in osteoblast. Further studies showed that C13 activated AMPK-dependent nicotinamide adenine dinucleotide phosphate (NADPH) pathway to inhibit Dex-induced reactive oxygen species (ROS) production in MC3T3-E1 cells and primary murine osteoblasts. Such effects by C13 were almost reversed by Compound C or AMPKα1 depletion/mutation. Together, these results suggest that C13 alleviates Dex-induced osteoblast cell death via activating AMPK signaling pathway. - Highlights: • Compound 13 (C13) attenuates dexamethasone (Dex)-induced osteoblast cell death. • C13-induced cytoprotective effect against Dex in osteoblasts requires AMPK activation. • Forced AMPK activation protects osteoblasts from Dex, nullifying C13's activities. • C13 increases NADPH activity and inhibits Dex-induced oxidative stress in osteoblasts.

CCR 20th Anniversary Commentary: MAPK/ERK Pathway Inhibition in Melanoma-Kinase Inhibition Redux.

Science.gov (United States)

Davar, Diwakar; Kirkwood, John M

2015-12-15

In the January 15, 2012, issue of Clinical Cancer Research, Kirkwood and colleagues published a study comparing the MEK inhibitor selumetinib with temozolomide in unselected metastatic melanoma. Although selumetinib did not improve survival or response, most responders had BRAF-activating mutations, and selumetinib has since demonstrated efficacy in BRAF-mutant melanoma. This study laid the groundwork for the evaluation of BRAF/MEK inhibitors in BRAF-mutant melanoma. ©2015 American Association for Cancer Research.

Clonorchis sinensis lysophospholipase A upregulates IL-25 expression in macrophages as a potential pathway to liver fibrosis.

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Zhou, Lina; Shi, Mengchen; Zhao, Lu; Lin, Zhipeng; Tang, Zeli; Sun, Hengchang; Chen, Tingjin; Lv, Zhiyue; Xu, Jin; Huang, Yan; Yu, Xinbing

2017-06-17

Liver fibrosis is an excessive wound-healing reaction that requires the participation of inflammatory cells and hepatic stellate cells (HSCs). The pathogenesis of liver fibrosis caused by viruses and alcohol has been well characterized, but the molecular mechanisms underlying liver fibrosis induced by the liver fluke Clonorchis sinensis are poorly understood. Lysophospholipase A (LysoPLA), which deacylates lysophospholipids, plays a critical role in mediating the virulence and pathogenesis of parasites and fungi; however, the roles of C. sinensis lysophospholipase A (CsLysoPLA) in C. sinensis-induced liver fibrosis remain unknown. A mouse macrophage cell line (RAW264.7) was cultured and treated with CsLysoPLA. IL-25 and members of its associated signaling pathway were detected by performing quantitative real-time PCR, Western blotting and immunofluorescent staining. A human hepatic stellate cell line (LX-2) was cultured and exposed to IL-25. LX-2 cell activation markers were examined via quantitative real-time PCR, Western blotting and immunofluorescent staining. Migration was analyzed in transwell plates. Treating RAW264.7 cells with CsLysoPLA significantly induced IL-25 expression. Elevated PKA, B-Raf, and ERK1/2 mRNA levels and phosphorylated B-Raf and ERK1/2 were detected in CsLysoPLA-stimulated RAW264.7 cells. The PKA inhibitor H-89 weakened B-Raf and ERK1/2 phosphorylation whereas the AKT activator SC79 attenuated ERK1/2 phosphorylation in RAW264.7 cells. Both H-89 and SC79 inhibited CsLysoPLA-induced IL-25 upregulation. In addition, stimulation of LX-2 cells with IL-25 upregulated the expression of mesenchymal cell markers, including α-smooth muscle actin (α-SMA) and collagen type I (Collagen-I), and promoted cell migration. CsLysoPLA activates HSCs by upregulating IL-25 in macrophages through the PKA-dependent B-Raf/ERK1/2 pathway and potentially promotes hepatic fibrosis during C. sinensis infection.

Effects of scallop shell extract on scopolamine-induced memory impairment and MK801-induced locomotor activity.

Science.gov (United States)

Hasegawa, Yasushi; Inoue, Tatsuro; Kawaminami, Satoshi; Fujita, Miho

2016-07-01

To evaluate the neuroprotective effects of the organic components of scallop shells (scallop shell extract) on memory impairment and locomotor activity induced by scopolamine or 5-methyl-10,11-dihydro-5H-dibenzo (a,d) cyclohepten-5,10-imine (MK801). Effect of the scallop shell extract on memory impairment and locomotor activity was investigated using the Y-maze test, the Morris water maze test, and the open field test. Scallop shell extract significantly reduced scopolamine-induced short-term memory impairment and partially reduced scopolamine-induced spatial memory impairment in the Morris water maze test. Scallop shell extract suppressed scopolamine-induced elevation of acetylcholine esterase activity in the cerebral cortex. Treatment with scallop shell extract reversed the increase in locomotor activity induced by scopolamine. Scallop shell extract also suppressed the increase in locomotor activity induced by MK801. Our results provide initial evidence that scallop shell extract reduces scopolamine-induced memory impairment and suppresses MK-801-induced hyperlocomotion. Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Contribution of Beta-HPV Infection and UV-Damage to Rapid-onset Cutaneous Squamous Cell Carcinoma during BRAF-inhibition Therapy

Science.gov (United States)

Cohen, Daniel N.; Lawson, Steven K.; Shaver, Aaron C.; Du, Liping; Nguyen, Harrison P.; He, Qin; Johnson, Douglas B.; Lumbang, Wilfred A.; Moody, Brent R.; Prescott, James L.; Chandra, Pranil K.; Boyd, Alan S.; Zwerner, Jeffrey P.; Robbins, Jason B.; Tyring, Stephen K.; Rady, Peter L.; Chappell, James D.; Shyr, Yu; Infante, Jeffrey R.; Sosman, Jeffrey A.

2015-01-01

Purpose BRAF-inhibition (BRAFi) therapy for advanced melanoma carries a high rate of secondary cutaneous squamous cell carcinoma (cSCC) and risk of other cancers. Ultraviolet (UV) radiation and α-genus human papillomavirus (HPV) are highly associated with SCC, but a novel role for β-genus HPV is suspected in BRAFi-cSCC. Cutaneous β-HPV may act in concert with host and environmental factors in BRAFi-cSCC. Experimental Design Primary BRAFi-cSCC tissue DNA isolated from patients receiving vemurafenib (Vem) or dabrafenib from two cancer centers was analyzed for the presence of cutaneous oncogenic viruses and host genetic mutations. Diagnostic specimens underwent consensus dermatopathology review. Clinical parameters for UV exposure and disease course were statistically analyzed in conjunction with histopathology. Results Twenty-nine patients contributed 69 BRAFi-cSCC lesions. BRAFi-cSCC had wart-like features (BRAFi-cSCC-WF) in 22% of specimens. During Vem therapy, BRAFi-cSCC-WF arose 11.6 weeks more rapidly than conventional-cSCC when controlled for gender and UV-exposure (p-value=0.03). Among all BRAFi-cSCC, β-genus HPV-17, HPV-38, HPV-111 were most frequently isolated and novel β-HPV genotypes were discovered (CTR, CRT-11, CRT-22). Sequencing revealed 63% of evaluated BRAFi-cSCCs harbored RAS mutations with PIK3CA, CKIT, ALK and EGFR mutations also detected. Conclusions We examined clinical, histopathologic, viral and genetic parameters in BRAFi-cSCC demonstrating rapid onset; wart-like histomorphology; β-HPV-17, HPV-38, and HPV-111 infection; UV damage; and novel ALK and CKIT mutations. Discovered β-HPV genotypes expand the spectrum of tumor-associated viruses. These findings enhance our understanding of factors cooperating with BRAF inhibition that accelerate keratinocyte oncogenesis as well as broaden the knowledge base of multifactorial mediators of cancer in general. PMID:25724524

Use of human tissue to assess the oncogenic activity of melanoma-associated mutations.

Science.gov (United States)

Chudnovsky, Yakov; Adams, Amy E; Robbins, Paul B; Lin, Qun; Khavari, Paul A

2005-07-01

Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Induction of Ras and Raf can be caused by active N-Ras and B-Raf mutants as well as by gene amplification. Activation of PI3K pathway components occurs by PTEN loss and by AKT3 amplification. Melanomas also commonly show impairment of the p16(INK4A)-CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16(INK4A) and ARF protein loss. Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification. In addition to ARF deletion, p53 pathway disruption can result from dominant negative TP53 mutations. TERT amplification also occurs in melanoma. The extent to which these mutations can induce human melanocytic neoplasia is unknown. Here we characterize pathways sufficient to generate human melanocytic neoplasia and show that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

Shock-induced electrical activity in polymeric solids. A mechanically induced bond scission model

International Nuclear Information System (INIS)

Graham, R.A.

1979-01-01

When polymeric solids are subjected to high-pressure shock loading, two anomalous electrical phenomena, shock-induced conduction and shock-induced polarization, are observed. The present paper proposes a model of mechanically induced bond scission within the shock front to account for the effects. An experimental study of shock-induced polarization in poly(pyromellitimide) (Vespel SP-1) is reported for shock compressions from 17 to 23% (pressures from 2.5 to 5.4 GPa). Poly(pyromellitimide) is found to be a strong generator of such polarization and the polarization is found to reflect an irreversible or highly hysteretic process. The present measurements are combined with prior measurements to establish a correlation between monomer structure and strength of shock-induced polarization; feeble signals are observed in the simpler monomer repeat units of poly(tetrafluoroethylene) and polyethylene while the strongest signals are observed in more complex monomers of poly(methyl methacrylate) and poly(pyromellitimide). It is also noted that there is an apparent correlation between shock-induced conduction and shock-induced polarization. Such shock-induced electrical activity is also found to be well correlated with the propensity for mechanical bond scission observed in experiments carried out in conventional mechanochemical studies. The bond scission model can account for characteristics observed for electrical activity in shock-loaded polymers and their correlation to monomer structure. Localization of elastic energy within the monomer repeat unit or along the main chain leads to the different propensities for bond scission and resulting shock-induced electrical activity

A cost-effectiveness analysis of trametinib plus dabrafenib as first-line therapy for metastatic BRAF V600-mutated melanoma in the Swiss setting.

Science.gov (United States)

Matter-Walstra, K; Braun, R; Kolb, C; Ademi, Z; Dummer, R; Pestalozzi, B C; Schwenkglenks, M

2015-12-01

The treatment of patients with metastatic melanomas that harbour BRAF V600E or V600K mutations with trametinib plus dabrafenib appears to be superior to treatment with vemurafenib alone. This treatment regimen is likely to become available in Switzerland in the near future. To determine the cost-effectiveness of trametinib plus dabrafenib. A Markov cohort simulation was conducted to model the clinical course of typical patients with metastatic melanoma. Information on response rates, clinical condition and follow-up treatments were derived and transition probabilities estimated based on the results of a clinical trial that compared treatment with trametinib plus dabrafenib vs. vemurafenib alone. Treatment with trametinib plus dabrafenib was estimated to cost an additional CHF199 647 (Swiss francs) on average and yield a gain of 0·52 quality-adjusted life years (QALYs), resulting in an incremental cost-effectiveness ratio of CHF385 603 per QALY. Probabilistic sensitivity analyses showed that a willingness-to-pay threshold of CHF100 000 per QALY would not be reached at the current US price of trametinib. The introduction of trametinib in Switzerland at US market prices for the treatment of metastatic BRAF V600-mutated melanoma with trametinib plus dabrafenib is unlikely to be cost-effective compared with vemurafenib monotherapy. A reduction in the total price of the combination therapy is required to achieve an acceptable cost-effectiveness ratio for this clinically promising treatment. © 2015 British Association of Dermatologists.

Constitutive activation of the ERK pathway in melanoma and skin melanocytes in Grey horses.

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Jiang, Lin; Campagne, Cécile; Sundström, Elisabeth; Sousa, Pedro; Imran, Saima; Seltenhammer, Monika; Pielberg, Gerli; Olsson, Mats J; Egidy, Giorgia; Andersson, Leif; Golovko, Anna

2014-11-21

Constitutive activation of the ERK pathway, occurring in the vast majority of melanocytic neoplasms, has a pivotal role in melanoma development. Different mechanisms underlie this activation in different tumour settings. The Grey phenotype in horses, caused by a 4.6 kb duplication in intron 6 of Syntaxin 17 (STX17), is associated with a very high incidence of cutaneous melanoma, but the molecular mechanism behind the melanomagenesis remains unknown. Here, we investigated the involvement of the ERK pathway in melanoma development in Grey horses. Grey horse melanoma tumours, cell lines and normal skin melanocytes were analyzed with help of indirect immunofluorescence and immunoblotting for the expression of phospho-ERK1/2 in comparison to that in non-grey horse and human counterparts. The mutational status of BRAF, RAS, GNAQ, GNA11 and KIT genes in Grey horse melanomas was determined by direct sequencing. The effect of RAS, RAF and PI3K/AKT pathways on the activation of the ERK signaling in Grey horse melanoma cells was investigated with help of specific inhibitors and immunoblotting. Individual roles of RAF and RAS kinases on the ERK activation were examined using si-RNA based approach and immunoblotting. We found that the ERK pathway is constitutively activated in Grey horse melanoma tumours and cell lines in the absence of somatic activating mutations in BRAF, RAS, GNAQ, GNA11 and KIT genes or alterations in the expression of the main components of the pathway. The pathway is mitogenic and is mediated by BRAF, CRAF and KRAS kinases. Importantly, we found high activation of the ERK pathway also in epidermal melanocytes, suggesting a general predisposition to melanomagenesis in these horses. These findings demonstrate that the presence of the intronic 4.6 kb duplication in STX17 is strongly associated with constitutive activation of the ERK pathway in melanocytic cells in Grey horses in the absence of somatic mutations commonly linked to the activation of this

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer.

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Paolo Cossu-Rocca

Full Text Available Triple Negative Breast Cancer (TNBC accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer.

Science.gov (United States)

Cossu-Rocca, Paolo; Orrù, Sandra; Muroni, Maria Rosaria; Sanges, Francesca; Sotgiu, Giovanni; Ena, Sara; Pira, Giovanna; Murgia, Luciano; Manca, Alessandra; Uras, Maria Gabriela; Sarobba, Maria Giuseppina; Urru, Silvana; De Miglio, Maria Rosaria

2015-01-01

Triple Negative Breast Cancer (TNBC) accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

Activation of Nrf2 protects against triptolide-induced hepatotoxicity.

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Jia Li

Full Text Available Triptolide, the major active component of Tripterygium wilfordii Hook f. (TWHF, has a wide range of pharmacological activities. However, the toxicities of triptolide, particularly the hepatotoxicity, limit its clinical application. The hepatotoxicity of triptolide has not been well characterized yet. The aim of this study was to investigate the role of NF-E2-related factor 2 (Nrf2 in triptolide-induced toxicity and whether activation of Nrf2 could protect against triptolide-induced hepatotoxicity. The results showed that triptolide caused oxidative stress and cell damage in HepG2 cells, and these toxic effects could be aggravated by Nrf2 knockdown or be counteracted by overexpression of Nrf2. Treatment with a typical Nrf2 agonist, sulforaphane (SFN, attenuated triptolide-induced liver dysfunction, structural damage, glutathione depletion and decrease in antioxidant enzymes in BALB/C mice. Moreover, the hepatoprotective effect of SFN on triptolide-induced liver injury was associated with the activation of Nrf2 and its downstream targets. Collectively, these results indicate that Nrf2 activation protects against triptolide-induced hepatotoxicity.

Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

International Nuclear Information System (INIS)

Nakatsu, Yusuke; Kotake, Yaichiro; Hino, Atsuko; Ohta, Shigeru

2008-01-01

AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release

EGFR Activation and Ultraviolet Light‐Induced Skin Carcinogenesis

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Taghrid B. El-Abaseri

2007-01-01

Full Text Available The epidermal growth factor receptor (EGFR regulates the proliferation of keratinocytes through multiple mechanisms that differ depending on the localization of the cell within the skin. Ultraviolet (UV irradiation, the main etiologic factor in the development of skin cancer, also activates the receptor. In this review, we discuss how the UV-induced activation of EGFR regulates the response of the skin to UV. UV-induced EGFR activation increases keratinocyte proliferation, suppresses apoptosis, and augments and accelerates epidermal hyperplasia in response to UV. Pharmacological inhibition of the UV-induced activation of EGFR in a genetically initiated mouse skin tumorigenesis model suppresses tumorigenesis and the activation of mitogen-activated protein (MAP kinases and phosphatidyl inositol-3-kinase (PI3K/AKT signaling pathways. EGFR has pleiotropic, complex, and cell-type-specific functions in cutaneous keratinocytes; suggesting that the receptor is an appropriate target for the development of molecularly targeted therapies for skin cancer and other pathologies.

Fisetin inhibits human melanoma cell invasion through promotion of mesenchymal to epithelial transition and by targeting MAPK and NFκB signaling pathways.

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Harish Chandra Pal

Full Text Available Malignant melanoma is responsible for approximately 75% of skin cancer-related deaths. BRAF plays an important role in regulating the mitogen-activated protein kinase (MAPK signaling cascade in melanoma with activating mutations in the serine/threonine kinase BRAF occurring in 60-70% of malignant melanomas. The BRAF-MEK-ERK (MAPK pathway is a key regulator of melanoma cell invasion. In addition, activation of NFκB via the MAPK pathway is regulated through MEK-induced activation of IKK. These pathways are potential targets for prevention and treatment of melanoma. In this study, we investigated the effect of fisetin, a phytochemical present in fruits and vegetables, on melanoma cell invasion and epithelial-mesenchymal transition, and delineated the underlying molecular mechanism. Treatment of multiple human malignant melanoma cell lines with fisetin (5-20 µM resulted in inhibition of cell invasion. BRAF mutated melanoma cells were more sensitive to fisetin treatment, and this was associated with a decrease in the phosphorylation of MEK1/2 and ERK1/2. In addition, fisetin inhibited the activation of IKK leading to a reduction in the activation of the NFκB signaling pathway. Treatment of cells with an inhibitor of MEK1/2 (PD98059 or of NFκB (caffeic acid phenethyl ester also reduced melanoma cell invasion. Furthermore, treatment of fisetin promoted mesenchymal to epithelial transition in melanoma cells, which was associated with a decrease in mesenchymal markers (N-cadherin, vimentin, snail and fibronectin and an increase in epithelial markers (E-cadherin and desmoglein. Employing three dimensional skin equivalents consisting of A375 cells admixed with normal human keratinocytes embedded onto a collagen-constricted fibroblast matrix, we found that treatment of fisetin reduced the invasive potential of melanoma cells into the dermis and increased the expression of E-cadherin with a concomitant decrease in vimentin. These results indicate that

Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

Science.gov (United States)

Pal, Harish Chandra; Sharma, Samriti; Strickland, Leah Ray; Katiyar, Santosh K.; Ballestas, Mary E.; Athar, Mohammad; Elmets, Craig A.; Afaq, Farrukh

2014-01-01

Malignant melanoma is responsible for approximately 75% of skin cancer-related deaths. BRAF plays an important role in regulating the mitogen-activated protein kinase (MAPK) signaling cascade in melanoma with activating mutations in the serine/threonine kinase BRAF occurring in 60–70% of malignant melanomas. The BRAF-MEK-ERK (MAPK) pathway is a key regulator of melanoma cell invasion. In addition, activation of NFκB via the MAPK pathway is regulated through MEK-induced activation of IKK. These pathways are potential targets for prevention and treatment of melanoma. In this study, we investigated the effect of fisetin, a phytochemical present in fruits and vegetables, on melanoma cell invasion and epithelial-mesenchymal transition, and delineated the underlying molecular mechanism. Treatment of multiple human malignant melanoma cell lines with fisetin (5–20 µM) resulted in inhibition of cell invasion. BRAF mutated melanoma cells were more sensitive to fisetin treatment, and this was associated with a decrease in the phosphorylation of MEK1/2 and ERK1/2. In addition, fisetin inhibited the activation of IKK leading to a reduction in the activation of the NFκB signaling pathway. Treatment of cells with an inhibitor of MEK1/2 (PD98059) or of NFκB (caffeic acid phenethyl ester) also reduced melanoma cell invasion. Furthermore, treatment of fisetin promoted mesenchymal to epithelial transition in melanoma cells, which was associated with a decrease in mesenchymal markers (N-cadherin, vimentin, snail and fibronectin) and an increase in epithelial markers (E-cadherin and desmoglein). Employing three dimensional skin equivalents consisting of A375 cells admixed with normal human keratinocytes embedded onto a collagen-constricted fibroblast matrix, we found that treatment of fisetin reduced the invasive potential of melanoma cells into the dermis and increased the expression of E-cadherin with a concomitant decrease in vimentin. These results indicate that fisetin

Cold suppresses agonist-induced activation of TRPV1.

Science.gov (United States)

Chung, M-K; Wang, S

2011-09-01

Cold therapy is frequently used to reduce pain and edema following acute injury or surgery such as tooth extraction. However, the neurobiological mechanisms of cold therapy are not completely understood. Transient receptor potential vanilloid 1 (TRPV1) is a capsaicin- and heat-gated nociceptive ion channel implicated in thermosensation and pathological pain under conditions of inflammation or injury. Although capsaicin-induced nociception, neuropeptide release, and ionic currents are suppressed by cold, it is not known if cold suppresses agonist-induced activation of recombinant TRPV1. We demonstrate that cold strongly suppressed the activation of recombinant TRPV1 by multiple agonists and capsaicin-evoked currents in trigeminal ganglia neurons under normal and phosphorylated conditions. Cold-induced suppression was partially impaired in a TRPV1 mutant that lacked heat-mediated activation and potentiation. These results suggest that cold-induced suppression of TRPV1 may share a common molecular basis with heat-induced potentiation, and that allosteric inhibition may contribute, in part, to the cold-induced suppression. We also show that combination of cold and a specific antagonist of TRPV1 can produce an additive suppression. Our results provide a mechanistic basis for cold therapy and may enhance anti-nociceptive approaches that target TRPV1 for managing pain under inflammation and tissue injury, including that from tooth extraction.

Activation of aryl hydrocarbon receptor reduces carbendazim-induced cell death

International Nuclear Information System (INIS)

Wei, Kuo-Liang; Chen, Fei-Yun; Lin, Chih-Yi; Gao, Guan-Lun; Kao, Wen-Ya; Yeh, Chi-Hui; Chen, Chang-Rong; Huang, Hao-Chun; Tsai, Wei-Ren; Jong, Koa-Jen; Li, Wan-Jung; Su, Jyan-Gwo Joseph

2016-01-01

Carbendazim inhibits microtubule assembly, thus blocking mitosis and inhibiting cancer cell proliferation. Accordingly, carbendazim is being explored as an anticancer drug. Data show that carbendazim increased mRNA and protein expressions and promoter activity of CYP1A1. In addition, carbendazim activated transcriptional activity of the aryl hydrocarbon response element, and induced nuclear translocation of the aryl hydrocarbon receptor (AhR), a sign the AhR is activated. Carbendazim-induced CYP1A1 expression was blocked by AhR antagonists, and was abolished in AhR signal-deficient cells. Results demonstrated that carbendazim activated the AhR, thereby stimulating CYP1A1 expression. In order to understand whether AhR-induced metabolic enzymes turn carbendazim into less-toxic metabolites, Hoechst 33342 staining to reveal carbendazim-induced nuclear changes and flow cytometry to reveal the subG 0 /G 1 population were applied to monitor carbendazim-induced cell apoptosis. Carbendazim induced less apoptosis in Hepa-1c1c7 cells than in AhR signal-deficient Hepa-1c1c7 mutant cells. Pretreatment with β-NF, an AhR agonist that highly induces CYP1A1 expression, decreased carbendazim-induced cell death. In addition, the lower the level of AhR was, the lower the vitality present in carbendazim-treated cells, including hepatoma cells and their derivatives with AhR RNA interference, also embryonic kidney cells, bladder carcinoma cells, and AhR signal-deficient Hepa-1c1c7 cells. In summary, carbendazim is an AhR agonist. The toxicity of carbendazim was lower in cells with the AhR signal. This report provides clues indicating that carbendazim is more potent at inducing cell death in tissues without than in those with the AhR signal, an important reference for applying carbendazim in cancer chemotherapy. - Highlights: • Carbendazim induced transcriptional activity of the aryl hydrocarbon response element. • Carbendazim induced nuclear translocation of the aryl hydrocarbon

Activation of aryl hydrocarbon receptor reduces carbendazim-induced cell death

Energy Technology Data Exchange (ETDEWEB)

Wei, Kuo-Liang [Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan, ROC (China); College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan, ROC (China); Chen, Fei-Yun; Lin, Chih-Yi [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Gao, Guan-Lun [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Department of Biological Resources, National Chiayi University, Chiayi, 60004, Taiwan, ROC (China); Kao, Wen-Ya [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Yeh, Chi-Hui [Department of Environmental Engineering, College of Engineering, Da-Yeh University, Dacun, Changhua 51591, Taiwan, ROC (China); Chen, Chang-Rong [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Huang, Hao-Chun [Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chang Gung Memorial Hospital, Chiayi 61363, Taiwan, ROC (China); Tsai, Wei-Ren [Division of Applied Toxicology, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Council of Agriculture, Executive Yuan, Taichung 41358, Taiwan, ROC (China); Jong, Koa-Jen [Department of Biological Resources, National Chiayi University, Chiayi, 60004, Taiwan, ROC (China); Li, Wan-Jung [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China); Su, Jyan-Gwo Joseph, E-mail: jgjsu@mail.ncyu.edu.tw [Department of Biochemical Science and Technology, National Chiayi University, Chiayi 60004, Taiwan, ROC (China)

2016-09-01

Carbendazim inhibits microtubule assembly, thus blocking mitosis and inhibiting cancer cell proliferation. Accordingly, carbendazim is being explored as an anticancer drug. Data show that carbendazim increased mRNA and protein expressions and promoter activity of CYP1A1. In addition, carbendazim activated transcriptional activity of the aryl hydrocarbon response element, and induced nuclear translocation of the aryl hydrocarbon receptor (AhR), a sign the AhR is activated. Carbendazim-induced CYP1A1 expression was blocked by AhR antagonists, and was abolished in AhR signal-deficient cells. Results demonstrated that carbendazim activated the AhR, thereby stimulating CYP1A1 expression. In order to understand whether AhR-induced metabolic enzymes turn carbendazim into less-toxic metabolites, Hoechst 33342 staining to reveal carbendazim-induced nuclear changes and flow cytometry to reveal the subG{sub 0}/G{sub 1} population were applied to monitor carbendazim-induced cell apoptosis. Carbendazim induced less apoptosis in Hepa-1c1c7 cells than in AhR signal-deficient Hepa-1c1c7 mutant cells. Pretreatment with β-NF, an AhR agonist that highly induces CYP1A1 expression, decreased carbendazim-induced cell death. In addition, the lower the level of AhR was, the lower the vitality present in carbendazim-treated cells, including hepatoma cells and their derivatives with AhR RNA interference, also embryonic kidney cells, bladder carcinoma cells, and AhR signal-deficient Hepa-1c1c7 cells. In summary, carbendazim is an AhR agonist. The toxicity of carbendazim was lower in cells with the AhR signal. This report provides clues indicating that carbendazim is more potent at inducing cell death in tissues without than in those with the AhR signal, an important reference for applying carbendazim in cancer chemotherapy. - Highlights: • Carbendazim induced transcriptional activity of the aryl hydrocarbon response element. • Carbendazim induced nuclear translocation of the aryl

GSK621 activates AMPK signaling to inhibit LPS-induced TNFα production

International Nuclear Information System (INIS)

Wu, Yong-hong; Li, Quan; Li, Ping; Liu, Bei

2016-01-01

LPS stimulation in macrophages/monocytes induces TNFα production. We here tested the potential effect of GSK621, a novel AMP-activated protein kinase (AMPK) activator, against the process. In RAW264.7 macrophages, murine bone marrow-derived macrophages (BMDMs), and chronic obstructive pulmonary disease (COPD) patients' monocytes, GSK621 significantly inhibited LPS-induced TNFα protein secretion and mRNA synthesis. Inhibition of AMPK, through AMPKα shRNA knockdown or dominant negative mutation (T172A), almost abolished GSK621's suppression on TNFα in RAW264.7 cells. Reversely, forced-expression of a constitutively-active AMPKα (T172D) mimicked GSK621 actions and reduced LPS-induced TNFα production. Molecularly, GSK621 suppressed LPS-induced reactive oxygen species (ROS) production and nuclear factor kappa B (NFκB) activation. In vivo, GSK621 oral administration inhibited LPS-induced TNFα production and endotoxin shock in mice. In summary, GSK621 activates AMPK signaling to inhibit LPS-induced TNFα production in macrophages/monocytes. - Highlights: • GSK621 inhibits LPS-induced TNFα production/expression in RAW264.7 cells and BMDMs. • GSK621 inhibits LPS-induced TNFα production/expression in COPD patients' PBMCs. • GSK621's inhibition on TNFα production by LPS requires AMPK activation. • GSK621 inhibits LPS-induced ROS production and NFκB activation, dependent on AMPK. • GSK621 oral administration inhibits LPS-induced TNFα production and endotoxin shock in mice.

Salicylate-induced abnormal activity in the inferior colliculus of rats.

Science.gov (United States)

Chen, G D; Jastreboff, P J

1995-02-01

The evaluation of the spontaneous activity of 471 units from the external nucleus of the IC revealed that salicylate induces an increase of the spontaneous activity and the emergence of a bursting type of activity longer than 4 spikes. For sharply tuned units, the affected cells were from the frequency range of 10-16 kHz, which corresponds to the behaviorally measured pitch of salicylate-induced tinnitus in rats. An exogenous calcium supplement, provided under the conditions shown to attenuate the behavioral manifestation of salicylate-induced tinnitus, abolished the modification of the spontaneous activity induced by salicylate. Finally, profound changes of activity were observed for cells not responding to contralateral sound. We propose that the observed long bursts of discharges represent tinnitus-related neuronal activity. The results are consistent with the hypothesis that GABA-mediated disinhibition is involved in the processing of tinnitus-related neuronal activity.

MAP2K1 and MAP3K1 mutations in langerhans cell histiocytosis

NARCIS (Netherlands)

Nelson, David S.; van Halteren, Astrid; Quispel, Willemijn T.; van den Bos, Cor; Bovée, Judith V. M. G.; Patel, Bhumi; Badalian-Very, Gayane; van Hummelen, Paul; Ducar, Matthew; Lin, Ling; MacConaill, Laura E.; Egeler, R. Maarten; Rollins, Barrett J.

2015-01-01

Langerhans cell histiocytosis (LCH) is now understood to be a neoplastic disease in which over 50% of cases have somatic activating mutations of BRAF. However, the extracellular signal-related (ERK) pathway is activated in all cases including those with wild type BRAF alleles. Here, we applied a

Effects of Parecoxib and Fentanyl on nociception-induced cortical activity

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Wang Ying-Wei

2010-01-01

Full Text Available Abstract Background Analgesics, including opioids and non-steroid anti-inflammatory drugs reduce postoperative pain. However, little is known about the quantitative effects of these drugs on cortical activity induced by nociceptive stimulation. The aim of the present study was to determine the neural activity in response to a nociceptive stimulus and to investigate the effects of fentanyl (an opioid agonist and parecoxib (a selective cyclooxygenase-2 inhibitor on this nociception-induced cortical activity evoked by tail pinch. Extracellular recordings (electroencephalogram and multi-unit signals were performed in the area of the anterior cingulate cortex while intracellular recordings were made in the primary somatosensory cortex. The effects of parecoxib and fentanyl on induced cortical activity were compared. Results Peripheral nociceptive stimulation in anesthetized rats produced an immediate electroencephalogram (EEG desynchronization resembling the cortical arousal (low-amplitude, fast-wave activity, while the membrane potential switched into a persistent depolarization state. The induced cortical activity was abolished by fentanyl, and the fentanyl's effect was reversed by the opioid receptor antagonist, naloxone. Parecoxib, on the other hand, did not significantly affect the neural activity. Conclusion Cortical activity was modulated by nociceptive stimulation in anesthetized rats. Fentanyl showed a strong inhibitory effect on the nociceptive-stimulus induced cortical activity while parecoxib had no significant effect.

BRAFV600E: implications for carcinogenesis and molecular therapy.

LENUS (Irish Health Repository)

Cantwell-Dorris, Emma R

2012-02-01

The mitogen-activated protein kinase (MAPK)\\/extracellular signal-regulated kinase (ERK) pathway is frequently mutated in human cancer. This pathway consists of a small GTP protein of the RAS family that is activated in response to extracellular signaling to recruit a member of the RAF kinase family to the cell membrane. Active RAF signals through MAP\\/ERK kinase to activate ERK and its downstream effectors to regulate a wide range of biological activities including cell differentiation, proliferation, senescence, and survival. Mutations in the v-raf murine sarcoma viral oncogenes homolog B1 (BRAF) isoform of the RAF kinase or KRAS isoform of the RAS protein are found as activating mutations in approximately 30% of all human cancers. The BRAF pathway has become a target of interest for molecular therapy, with promising results emerging from clinical trials. Here, the role of the most common BRAF mutation BRAF(V600E) in human carcinogenesis is investigated through a review of the literature, with specific focus on its role in melanoma, colorectal, and thyroid cancers and its potential as a therapeutic target.

Absence of the BRAF mutation in HBME1+ and CK19+ atypical cell clusters in Hashimoto thyroiditis: supportive evidence against preneoplastic change.

Science.gov (United States)

Nasr, Michel R; Mukhopadhyay, Sanjay; Zhang, Shengle; Katzenstein, Anna-Luise A

2009-12-01

An association between Hashimoto thyroiditis and papillary thyroid carcinoma has been postulated for decades. We undertook this study to identify potential precursors of papillary thyroid carcinoma in Hashimoto thyroiditis using a combination of morphologic, immunohistochemical, and molecular techniques. For the study, samples from 59 cases of Hashimoto thyroiditis were stained with antibodies to HBME1 and cytokeratin (CK)19. Tiny HBME1+ and CK19+ atypical cell clusters were identified and analyzed for the BRAF mutation by the colorimetric Mutector assay and allele-specific polymerase chain reaction. HBME1+ and CK19+ atypical cell clusters were identified in 12 (20%) of 59 cases. The minute size (Hashimoto thyroiditis.

Somatic mutations in histiocytic sarcoma identified by next generation sequencing.

Science.gov (United States)

Liu, Qingqing; Tomaszewicz, Keith; Hutchinson, Lloyd; Hornick, Jason L; Woda, Bruce; Yu, Hongbo

2016-08-01

Histiocytic sarcoma is a rare malignant neoplasm of presumed hematopoietic origin showing morphologic and immunophenotypic evidence of histiocytic differentiation. Somatic mutation importance in the pathogenesis or disease progression of histiocytic sarcoma was largely unknown. To identify somatic mutations in histiocytic sarcoma, we studied 5 histiocytic sarcomas [3 female and 2 male patients; mean age 54.8 (20-72), anatomic sites include lymph node, uterus, and pleura] and matched normal tissues from each patient as germ line controls. Somatic mutations in 50 "Hotspot" oncogenes and tumor suppressor genes were examined using next generation sequencing. Three (out of five) histiocytic sarcoma cases carried somatic mutations in BRAF. Among them, G464V [variant frequency (VF) of 43.6 %] and G466R (VF of 29.6 %) located at the P loop potentially interfere with the hydrophobic interaction between P and activating loops and ultimately activation of BRAF. Also detected was BRAF somatic mutation N581S (VF of 7.4 %), which was located at the catalytic loop of BRAF kinase domain: its role in modifying kinase activity was unclear. A similar mutational analysis was also performed on nine acute monocytic/monoblastic leukemia cases, which did not identify any BRAF somatic mutations. Our study detected several BRAF mutations in histiocytic sarcomas, which may be important in understanding the tumorigenesis of this rare neoplasm and providing mechanisms for potential therapeutical opportunities.

Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

Science.gov (United States)

Trotta, Andrew P; Gelles, Jesse D; Serasinghe, Madhavika N; Loi, Patrick; Arbiser, Jack L; Chipuk, Jerry E

2017-07-14

The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAF V600E ), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAF V600E is pharmacologically inhibited by targeted therapies ( e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAF V600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G 1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

Methylphenidate Actively Induces Emergence from General Anesthesia

Science.gov (United States)

Solt, Ken; Cotten, Joseph F.; Cimenser, Aylin; Wong, Kin F.K.; Chemali, Jessica J.; Brown, Emery N.

2011-01-01

Background Although accumulating evidence suggests that arousal pathways in the brain play important roles in emergence from general anesthesia, the roles of monoaminergic arousal circuits are unclear. In this study we tested the hypothesis that methylphenidate (an inhibitor of dopamine and norepinephrine transporters) induces emergence from isoflurane anesthesia. Methods Using adult rats we tested the effect of methylphenidate IV on time to emergence from isoflurane anesthesia. We then performed experiments to test separately for methylphenidate-induced changes in arousal and changes in minute ventilation. A dose-response study was performed to test for methylphenidate–induced restoration of righting during continuous isoflurane anesthesia. Surface electroencephalogram recordings were performed to observe neurophysiological changes. Plethysmography recordings and arterial blood gas analysis were performed to assess methylphenidate-induced changes in respiratory function. Droperidol IV was administered to test for inhibition of methylphenidate's actions. Results Methylphenidate decreased median time to emergence from 280 to 91 s. The median difference in time to emergence without compared to with methylphenidate was 200 [155, 331] s (median, [95% confidence interval]). During continuous inhalation of isoflurane, methylphenidate induced return of righting in a dose-dependent manner, induced a shift in electroencephalogram power from delta to theta, and induced an increase in minute ventilation. Administration of droperidol (0.5 mg/kg IV) prior to methylphenidate (5 mg/kg IV) largely inhibited methylphenidate-induced emergence behavior, electroencephalogram changes, and changes in minute ventilation. Conclusions Methylphenidate actively induces emergence from isoflurane anesthesia by increasing arousal and respiratory drive, possibly through activation of dopaminergic and adrenergic arousal circuits. Our findings suggest that methylphenidate may be clinically

Ethanol-Induced Neurodegeneration and Glial Activation in the Developing Brain

Directory of Open Access Journals (Sweden)

Mariko Saito

2016-08-01

Full Text Available Ethanol induces neurodegeneration in the developing brain, which may partially explain the long-lasting adverse effects of prenatal ethanol exposure in fetal alcohol spectrum disorders (FASD. While animal models of FASD show that ethanol-induced neurodegeneration is associated with glial activation, the relationship between glial activation and neurodegeneration has not been clarified. This review focuses on the roles of activated microglia and astrocytes in neurodegeneration triggered by ethanol in rodents during the early postnatal period (equivalent to the third trimester of human pregnancy. Previous literature indicates that acute binge-like ethanol exposure in postnatal day 7 (P7 mice induces apoptotic neurodegeneration, transient activation of microglia resulting in phagocytosis of degenerating neurons, and a prolonged increase in glial fibrillary acidic protein-positive astrocytes. In our present study, systemic administration of a moderate dose of lipopolysaccharides, which causes glial activation, attenuates ethanol-induced neurodegeneration. These studies suggest that activation of microglia and astrocytes by acute ethanol in the neonatal brain may provide neuroprotection. However, repeated or chronic ethanol can induce significant proinflammatory glial reaction and neurotoxicity. Further studies are necessary to elucidate whether acute or sustained glial activation caused by ethanol exposure in the developing brain can affect long-lasting cellular and behavioral abnormalities observed in the adult brain.

DA-Raf-Mediated Suppression of the Ras--ERK Pathway Is Essential for TGF-β1-Induced Epithelial-Mesenchymal Transition in Alveolar Epithelial Type 2 Cells.

Science.gov (United States)

Watanabe-Takano, Haruko; Takano, Kazunori; Hatano, Masahiko; Tokuhisa, Takeshi; Endo, Takeshi

2015-01-01

Myofibroblasts play critical roles in the development of idiopathic pulmonary fibrosis by depositing components of extracellular matrix. One source of lung myofibroblasts is thought to be alveolar epithelial type 2 cells that undergo epithelial-mesenchymal transition (EMT). Rat RLE-6TN alveolar epithelial type 2 cells treated with transforming growth factor-β1 (TGF-β1) are converted into myofibroblasts through EMT. TGF-β induces both canonical Smad signaling and non-canonical signaling, including the Ras-induced ERK pathway (Raf-MEK-ERK). However, the signaling mechanisms regulating TGF-β1-induced EMT are not fully understood. Here, we show that the Ras-ERK pathway negatively regulates TGF-β1-induced EMT in RLE-6TN cells and that DA-Raf1 (DA-Raf), a splicing isoform of A-Raf and a dominant-negative antagonist of the Ras-ERK pathway, plays an essential role in EMT. Stimulation of the cells with fibroblast growth factor 2 (FGF2), which activated the ERK pathway, prominently suppressed TGF-β1-induced EMT. An inhibitor of MEK, but not an inhibitor of phosphatidylinositol 3-kinase, rescued the TGF-β1-treated cells from the suppression of EMT by FGF2. Overexpression of a constitutively active mutant of a component of the Ras-ERK pathway, i.e., H-Ras, B-Raf, or MEK1, interfered with EMT. Knockdown of DA-Raf expression with siRNAs facilitated the activity of MEK and ERK, which were only weakly and transiently activated by TGF-β1. Although DA-Raf knockdown abrogated TGF-β1-induced EMT, the abrogation of EMT was reversed by the addition of the MEK inhibitor. Furthermore, DA-Raf knockdown impaired the TGF-β1-induced nuclear translocation of Smad2, which mediates the transcription required for EMT. These results imply that intrinsic DA-Raf exerts essential functions for EMT by antagonizing the TGF-β1-induced Ras-ERK pathway in RLE-6TN cells.

Unusual acute and delayed skin reactions during and after whole-brain radiotherapy in combination with the BRAF inhibitor vemurafenib. Two case reports

Energy Technology Data Exchange (ETDEWEB)

Schulze, B.; Roedel, C.; Weiss, C. [Johann Wolfgang Goethe-University Hospital, Department of Radiation Oncology, Frankfurt am Main (Germany); Meissner, M.; Wolter, M. [Johann Wolfgang Goethe-University Hospital, Department of Dermatology, Frankfurt am Main (Germany)

2014-02-15

Besides radiotherapy (RT) and surgery, the introduction of BRAF inhibitors like vemurafenib has provided new opportunities for treatment of patients with metastasized malignant melanomas. RT and vemurafenib are being increasingly used concurrently, although little is known about the potential side effects of this combination. Vemurafenib is known to cause severe cutaneous skin reactions such as phototoxicity and evidence is accumulating that RT may further enhance these side effects. We report two cases of unusual skin reactions occurring during and after treatment with a combination of vemurafenib and whole-brain irradiation in patients with cerebral metastases arising from malignant melanomas. One case report describes excessive acute radiodermatitis which arose during whole-brain irradiation in combination with vemurafenib. The second describes a late skin reaction occurring approximately 30 days after completion of RT. These two case reports show that combination of both treatment modalities is possible, but requires close monitoring of patients and good interdisciplinary collaboration. (orig.) [German] Neben der Strahlentherapie und Chirurgie stellt die Einfuehrung von BRAF-Inhibitoren wie Vemurafenib eine neue Moeglichkeit zur Behandlung von metastasierten malignen Melanomen dar und immer haeufiger kommt eine Kombination aus Strahlentherapie und Vemurafenib zum Einsatz. Bislang ist wenig bekannt ueber potentielle Nebenwirkungen, die sich aus einer Kombination beider Therapieoptionen ergeben koennen. Vemurafenib kann zu schweren kutanen Nebenwirkungen wie z. B. Phototoxizitaet fuehren und es haeufen sich Hinweise, dass die Strahlentherapie diese Nebenwirkungen verstaerken kann. Wir berichten ueber zwei Faelle ungewoehnlicher Hautreaktionen waehrend und nach einer Ganzhirnbestrahlung in Kombination mit Vemurafenib. Ein Fall beschreibt eine akute und ueberschiessende Radiodermatitis unter fortlaufender Radiotherapie und der andere Fall beschreibt eine spaete

Histamine induces microglia activation and dopaminergic neuronal toxicity via H1 receptor activation.

Science.gov (United States)

Rocha, Sandra M; Saraiva, Tatiana; Cristóvão, Ana C; Ferreira, Raquel; Santos, Tiago; Esteves, Marta; Saraiva, Cláudia; Je, Goun; Cortes, Luísa; Valero, Jorge; Alves, Gilberto; Klibanov, Alexander; Kim, Yoon-Seong; Bernardino, Liliana

2016-06-04

Histamine is an amine widely known as a peripheral inflammatory mediator and as a neurotransmitter in the central nervous system. Recently, it has been suggested that histamine acts as an innate modulator of microglial activity. Herein, we aimed to disclose the role of histamine in microglial phagocytic activity and reactive oxygen species (ROS) production and to explore the consequences of histamine-induced neuroinflammation in dopaminergic (DA) neuronal survival. The effect of histamine on phagocytosis was assessed both in vitro by using a murine N9 microglial cell line and primary microglial cell cultures and in vivo. Cells were exposed to IgG-opsonized latex beads or phosphatidylserine (PS) liposomes to evaluate Fcγ or PS receptor-mediated microglial phagocytosis, respectively. ROS production and protein levels of NADPH oxidases and Rac1 were assessed as a measure of oxidative stress. DA neuronal survival was evaluated in vivo by counting the number of tyrosine hydroxylase-positive neurons in the substantia nigra (SN) of mice. We found that histamine triggers microglial phagocytosis via histamine receptor 1 (H1R) activation and ROS production via H1R and H4R activation. By using apocynin, a broad NADPH oxidase (Nox) inhibitor, and Nox1 knockout mice, we found that the Nox1 signaling pathway is involved in both phagocytosis and ROS production induced by histamine in vitro. Interestingly, both apocynin and annexin V (used as inhibitor of PS-induced phagocytosis) fully abolished the DA neurotoxicity induced by the injection of histamine in the SN of adult mice in vivo. Blockade of H1R protected against histamine-induced Nox1 expression and death of DA neurons in vivo. Overall, our results highlight the relevance of histamine in the modulation of microglial activity that ultimately may interfere with neuronal survival in the context of Parkinson's disease (PD) and, eventually, other neurodegenerative diseases which are accompanied by microglia-induced

Hyperplastic polyps of the colon and rectum - reclassification, BRAF and KRAS status in index polyps and subsequent colorectal carcinoma

DEFF Research Database (Denmark)

Janjua, Huma Gul Rehana; Høgdall, Estrid; Linnemann, Dorte

2015-01-01

(THP), sessile serrated lesions (SSL), and other lesions. All patients were confirmed in the Danish National Pathology Database for the occurrence of metachronous polyps/adenomas, colorectal cancer (CRC), and other gastrointestinal malignancies. Molecular pathology of the CRC were characterized...... and correlated with the index lesion. In total, 591 HP biopsy specimens were obtained from 480 patients. The lesions were reclassified as: 358 THP, 109 SSL, 35 TA, 81 unspecified non-neoplastic lesions, four traditional serrated adenoma, and 4 SSL with cytological dysplasia. Seven patients developed CRC...... in the follow-up period (1 patient had SSL, 4 had THP, and 2 had unspecified non-neoplastic lesions). Ten patients developed other gastrointestinal malignancies. The patient with SSL as index lesions who developed CRC harbored V600E BRAF mutation in both index lesion and the carcinoma. Sixteen percent...

Misconceptions about mirror-induced motor cortex activation.

NARCIS (Netherlands)

Praamstra, P.; Torney, L.; Rawle, C.J.; Miall, R.C.

2011-01-01

Observation of self-produced hand movements through a mirror, creating an illusion of the opposite hand moving, was recently reported to induce ipsilateral motor cortex activation, that is, motor cortex activation for the hand in rest. The reported work goes far beyond earlier work on motor cortex

Gastroprotective activity of polysaccharide from Hericium erinaceus against ethanol-induced gastric mucosal lesion and pylorus ligation-induced gastric ulcer, and its antioxidant activities.

Science.gov (United States)

Wang, Xiao-Yin; Yin, Jun-Yi; Zhao, Ming-Ming; Liu, Shi-Yu; Nie, Shao-Ping; Xie, Ming-Yong

2018-04-15

The gastroprotective activity of Hericium erinaceus polysaccharide was investigated in rats. The antioxidant activities were also evaluated. Pre-treatment of polysaccharide could reduce ethanol-induced gastric mucosal lesion and pylorus ligation-induced gastric ulcer. The polysaccharide exhibited scavenging activities of 1, 1-diphenyl-2-picryl-hydrozyl and hydroxyl radicals, and ferrous ion-chelating ability. In the pylorus ligation-induced model, gastric secretions (volume of gastric juice, gastric acid, pepsin and mucus) of ulcer rats administrated with polysaccharide were regulated. Levels of tumor necrosis factor-α and interleukins-1β in serum, and myeloperoxidase activity of gastric tissue were reduced, while antioxidant status of gastric tissue was improved. Defensive factors (nitric oxide, prostaglandin E2, epidermal growth factor) in gastric tissue were increased. These results indicate that Hericium erinaceus polysaccharide possess gastroprotective activity, and the possible mechanisms are related to its regulations of gastric secretions, improvements of anti-inflammatory and antioxidant status, as well as increments of defensive factors releases. Copyright © 2018 Elsevier Ltd. All rights reserved.

Activity deprivation induces neuronal cell death: mediation by tissue-type plasminogen activator.

Directory of Open Access Journals (Sweden)

Eldi Schonfeld-Dado

Full Text Available Spontaneous activity is an essential attribute of neuronal networks and plays a critical role in their development and maintenance. Upon blockade of activity with tetrodotoxin (TTX, neurons degenerate slowly and die in a manner resembling neurodegenerative diseases-induced neuronal cell death. The molecular cascade leading to this type of slow cell death is not entirely clear. Primary post-natal cortical neurons were exposed to TTX for up to two weeks, followed by molecular, biochemical and immunefluorescence analysis. The expression of the neuronal marker, neuron specific enolase (NSE, was down-regulated, as expected, but surprisingly, there was a concomitant and striking elevation in expression of tissue-type plasminogen activator (tPA. Immunofluorescence analysis indicated that tPA was highly elevated inside affected neurons. Transfection of an endogenous tPA inhibitor, plasminogen activator inhibitor-1 (PAI-1, protected the TTX-exposed neurons from dying. These results indicate that tPA is a pivotal player in slowly progressing activity deprivation-induced neurodegeneration.

NOX2-Induced Activation of Arginase and Diabetes-Induced Retinal Endothelial Cell Senescence

Directory of Open Access Journals (Sweden)

Modesto Rojas

2017-06-01

Full Text Available Increases in reactive oxygen species (ROS and decreases in nitric oxide (NO have been linked to vascular dysfunction during diabetic retinopathy (DR. Diabetes can reduce NO by increasing ROS and by increasing activity of arginase, which competes with nitric oxide synthase (NOS for their commons substrate l-arginine. Increased ROS and decreased NO can cause premature endothelial cell (EC senescence leading to defective vascular repair. We have previously demonstrated the involvement of NADPH oxidase 2 (NOX2-derived ROS, decreased NO and overactive arginase in DR. Here, we investigated their impact on diabetes-induced EC senescence. Studies using diabetic mice and retinal ECs treated with high glucose or H2O2 showed that increases in ROS formation, elevated arginase expression and activity, and decreased NO formation led to premature EC senescence. NOX2 blockade or arginase inhibition prevented these effects. EC senescence was also increased by inhibition of NOS activity and this was prevented by treatment with a NO donor. These results indicate that diabetes/high glucose-induced activation of arginase and decreases in NO bioavailability accelerate EC senescence. NOX2-generated ROS contribute importantly to this process. Blockade of NOX2 or arginase represents a strategy to prevent diabetes-induced premature EC senescence by preserving NO bioavailability.

Use of Human Tissue to Assess the Oncogenic Activity of Melanoma-Associated Mutations

OpenAIRE

Chudnovsky, Yakov; Adams, Amy E.; Robbins, Paul B.; Lin, Qun; Khavari, Paul A.

2005-01-01

Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence1,2. These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Ras and Raf induction can occur via active N-Ras and B-Raf mutants as well as by gene amplification3–5. Activation of PI3K pathway components occurs by PTEN loss and by AKT amplification6–8. Melanomas also commonly display impairment of p16INK4A-CDK4-Rb and ARF-HDM2-p53 tumor s...

Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells

NARCIS (Netherlands)

Staszewski, Ori; Baker, Richard E.; Ucher, Anna J.; Martier, Raygene; Stavnezer, Janet; Guikema, Jeroen E. J.

2011-01-01

After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading

Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

Energy Technology Data Exchange (ETDEWEB)

Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

2014-08-01

Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

International Nuclear Information System (INIS)

Nagata, Yosuke; Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

2014-01-01

Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

The PANDA study: a randomized phase II study of first-line FOLFOX plus panitumumab versus 5FU plus panitumumab in RAS and BRAF wild-type elderly metastatic colorectal cancer patients.

Science.gov (United States)

Battaglin, Francesca; Schirripa, Marta; Buggin, Federica; Pietrantonio, Filippo; Morano, Federica; Boscolo, Giorgia; Tonini, Giuseppe; Lutrino, Eufemia Stefania; Lucchetti, Jessica; Salvatore, Lisa; Passardi, Alessandro; Cremolini, Chiara; Arnoldi, Ermenegildo; Scartozzi, Mario; Pella, Nicoletta; Boni, Luca; Bergamo, Francesca; Zagonel, Vittorina; Loupakis, Fotios; Lonardi, Sara

2018-01-25

Few data are available regarding the treatment of metastatic colorectal cancer elderly patients with anti-EGFR agents in combination with chemotherapy. FOLFOX plus panitumumab is a standard first-line option for RAS wild-type metastatic colorectal cancer. Slight adjustments in chemo-dosage are commonly applied in clinical practice to elderly patients, but those modified schedules have never been prospectively tested. Clinical definition of elderly (≥70 years old) patients that may deserve a more or less intensive combination therapy is still debated. Several geriatric screening tools have been developed to predict survival and risk of toxicity from treatment. Among those, the G8 screening tool has been tested in cancer patients showing the strongest prognostic value for overall survival, while the CRASH score can stratify patients according to an estimated risk of treatment-related toxicities. The PANDA study is a prospective, open-label, multicenter, randomized phase II trial of first-line therapy with panitumumab in combination with dose-adjusted FOLFOX or with 5-fluorouracil monotherapy, in previously untreated elderly patients (≥70 years) with RAS and BRAF wild-type unresectable metastatic colorectal cancer. RAS and BRAF analyses are centralized. Geriatric assessment by means of G8 and CRASH score is planned at baseline and G8 will be re-evaluated at disease progression. The primary endpoint is duration of progression-free survival in both arms. Secondary endpoints include prospective evaluation of the prognostic role of G8 score and the correlation of CRASH risk categories with toxicity. The PANDA study aims at exploring safety and efficacy of panitumumab in combination with FOLFOX or with 5FU/LV in elderly patients affected by RAS and BRAF wild-type metastatic colorectal cancer, to identify the most promising treatment strategy in this setting. Additionally, this is the first trial in which the prognostic role of the G8 score will be prospectively

Obesity-induced vascular inflammation involves elevated arginase activity.

Science.gov (United States)

Yao, Lin; Bhatta, Anil; Xu, Zhimin; Chen, Jijun; Toque, Haroldo A; Chen, Yongjun; Xu, Yimin; Bagi, Zsolt; Lucas, Rudolf; Huo, Yuqing; Caldwell, Ruth B; Caldwell, R William

2017-11-01

Obesity-induced vascular dysfunction involves pathological remodeling of the visceral adipose tissue (VAT) and increased inflammation. Our previous studies showed that arginase 1 (A1) in endothelial cells (ECs) is critically involved in obesity-induced vascular dysfunction. We tested the hypothesis that EC-A1 activity also drives obesity-related VAT remodeling and inflammation. Our studies utilized wild-type and EC-A1 knockout (KO) mice made obese by high-fat/high-sucrose (HFHS) diet. HFHS diet induced increases in body weight, fasting blood glucose, and VAT expansion. This was accompanied by increased arginase activity and A1 expression in vascular ECs and increased expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in both VAT and ECs. HFHS also markedly increased circulating inflammatory monocytes and VAT infiltration by inflammatory macrophages, while reducing reparative macrophages. Additionally, adipocyte size and fibrosis increased and capillary density decreased in VAT. These effects of HFHS, except for weight gain and hyperglycemia, were prevented or reduced in mice lacking EC-A1 or treated with the arginase inhibitor 2-( S )-amino-6-boronohexanoic acid (ABH). In mouse aortic ECs, exposure to high glucose (25 mM) and Na palmitate (200 μM) reduced nitric oxide production and increased A1, TNF-α, VCAM-1, ICAM-1, and MCP-1 mRNA, and monocyte adhesion. Knockout of EC-A1 or ABH prevented these effects. HFHS diet-induced VAT inflammation is mediated by EC-A1 expression/activity. Limiting arginase activity is a possible therapeutic means of controlling obesity-induced vascular and VAT inflammation.

Investigating the Causes of Chronic Itch: New Advances Could Bring Relief

Science.gov (United States)

... focuses on nerve cell involvement in chronic itch. His team was interested in a protein called BRAF, which is known to help convey pain signals from sensory nerves in the skin to the brain. Activated BRAF (represented in red), when introduced into ...

Detection of up to 65% of Precancerous Lesions of the Human Colon and Rectum by Mutation Analysis of APC, K-Ras, B-Raf and CTNNB1

International Nuclear Information System (INIS)

Schneider, Mandy; Scholtka, Bettina; Gottschalk, Uwe; Faiss, Siegbert; Schatz, Daniela; Berghof-Jäger, Kornelia; Steinberg, Pablo

2010-01-01

In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form. CTNNB1 and B-Raf were screened by PCR-single-strand conformation polymorphism analysis, K-Ras by restriction fragment length polymorphism analysis and the APC gene mutation cluster region (codons 1243–1567) by direct DNA sequencing. APC mutations were only detected in 10% of the serrated lesions but in 34% of the adenomas. Twenty percent of the serrated lesions and 14% of the adenomas carried a mutated K-Ras. B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas. CTNNB1 was altered in 12% of the adenomas, but not in serrated lesions. By using the above gene marker panel it could be shown that 65% of the serrated lesions and 61% of the adenomas carried at least one of the four genes in a mutated form. Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer

Detection of up to 65% of Precancerous Lesions of the Human Colon and Rectum by Mutation Analysis of APC, K-Ras, B-Raf and CTNNB1

Energy Technology Data Exchange (ETDEWEB)

Schneider, Mandy; Scholtka, Bettina, E-mail: scholtka@uni-potsdam.de [Chair of Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam, Arthur- Scheunert-Allee 114-116, 14558 Nuthetal (Germany); Gottschalk, Uwe [Maria Heimsuchung Caritas-Klinik Pankow, Breite Straße 46/47, 13187 Berlin (Germany); Faiss, Siegbert [III. Medizinische Abteilung - Gastroenterologie und Hepatologie, Asklepios Klinik Barmbek, Rubenkamp 220, 22291 Hamburg (Germany); Schatz, Daniela; Berghof-Jäger, Kornelia [BIOTECON Diagnostics GmbH, Hermannswerder Haus 17, 14473 Potsdam (Germany); Steinberg, Pablo, E-mail: scholtka@uni-potsdam.de [Chair of Nutritional Toxicology, Institute of Nutritional Science, University of Potsdam, Arthur- Scheunert-Allee 114-116, 14558 Nuthetal (Germany); Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover (Germany)

2010-12-29

In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form. CTNNB1 and B-Raf were screened by PCR-single-strand conformation polymorphism analysis, K-Ras by restriction fragment length polymorphism analysis and the APC gene mutation cluster region (codons 1243–1567) by direct DNA sequencing. APC mutations were only detected in 10% of the serrated lesions but in 34% of the adenomas. Twenty percent of the serrated lesions and 14% of the adenomas carried a mutated K-Ras. B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas. CTNNB1 was altered in 12% of the adenomas, but not in serrated lesions. By using the above gene marker panel it could be shown that 65% of the serrated lesions and 61% of the adenomas carried at least one of the four genes in a mutated form. Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer.

EPHA2 is a mediator of vemurafenib resistance and a novel therapeutic target in melanoma.

Science.gov (United States)

Miao, Benchun; Ji, Zhenyu; Tan, Li; Taylor, Michael; Zhang, Jianming; Choi, Hwan Geun; Frederick, Dennie T; Kumar, Raj; Wargo, Jennifer A; Flaherty, Keith T; Gray, Nathanael S; Tsao, Hensin

2015-03-01

BRAF(V600E) is the most common oncogenic lesion in melanoma and results in constitutive activation of the MAPK pathway and uncontrolled cell growth. Selective BRAF inhibitors such as vemurafenib have been shown to neutralize oncogenic signaling, restrain cellular growth, and improve patient outcome. Although several mechanisms of vemurafenib resistance have been described, directed solutions to overcome these resistance lesions are still lacking. Herein, we found that vemurafenib resistance can be (i) mediated by EPHA2, a member of the largest receptor tyrosine kinases (RTK) subfamily erythropoietin-producing hepatocellular (EPH) receptors, and (ii) associated with a greater phenotypic dependence on EPHA2. Furthermore, we developed a series of first-in-class EPHA2 inhibitors and show that these new compounds potently induce apoptosis, suppress viability, and abrogate tumorigenic growth of melanoma cells, including those that are resistant to vemurafenib. These results provide proof of concept that RTK-guided growth, and therapeutic resistance, can be prospectively defined and selectively targeted. In this study, we show that resistance to selective BRAF inhibitors can be mediated by the RTK EPHA2. Furthermore, direct targeting of EPHA2 can successfully suppress melanoma growth and mitigate therapeutic resistance. ©2014 American Association for Cancer Research.

Dihydrotestosterone Potentiates EGF-Induced ERK Activation by Inducing SRC in Fetal Lung Fibroblasts

Science.gov (United States)

Smith, Susan M.; Murray, Sandy; Pham, Lucia D.; Minoo, Parviz; Nielsen, Heber C.

2014-01-01

Lung maturation is regulated by interactions between mesenchymal and epithelial cells, and is delayed by androgens. Fibroblast–Type II cell communications are dependent on extracellular signal-regulated kinases (ERK) 1/2 activation by the ErbB receptor ligands epidermal growth factor (EGF), transforming growth factor (TGF)-α, and neuregulin (Nrg). In other tissues, dihydrotestosterone (DHT) has been shown to activate SRC by a novel nontranscriptional mechanism, which phosphorylates EGF receptors to potentiate EGF-induced ERK1/2 activation. This study sought to determine if DHT potentiates EGFR signaling by a nontranscriptional mechanism. Embryonic day (E)17 fetal lung cells were isolated from dams treated with or without DHT since E12. Cells were exposed to 30 ng/ml DHT for periods of 30 minutes to 3 days before being stimulated with 100 ng/ml EGF, TGF-α, or Nrg for up to 30 minutes. Lysates were immunoblotted for ErbB and SRC pathway signaling intermediates. DHT increased ERK1/2 activation by EGF, TGF-α, and Nrg in fibroblasts and Type II cells. Characterization in fibroblasts showed that potentiation of the EGF pathway was significant after 60 minutes of DHT exposure and persisted in the presence of the translational inhibitor cycloheximide. SRC and EGF receptor phosphorylation was increased by DHT, as was EGF-induced SHC1 phosphorylation and subsequent association with GRB2. Finally, SRC silencing, SRC inhibition with PP2, and overexpression of a dominant-negative SRC each prevented DHT from increasing EGF-induced ERK1/2 phosphorylation. These results suggest that DHT activates SRC to potentiate the signaling pathway leading from the EGF receptor to ERK activation in primary fetal lung fibroblasts. PMID:24484548

Epstein-Barr Virus Lytic Reactivation Activates B Cells Polyclonally and Induces Activation-Induced Cytidine Deaminase Expression: A Mechanism Underlying Autoimmunity and Its Contribution to Graves' Disease.

Science.gov (United States)

Nagata, Keiko; Kumata, Keisuke; Nakayama, Yuji; Satoh, Yukio; Sugihara, Hirotsugu; Hara, Sayuri; Matsushita, Michiko; Kuwamoto, Satoshi; Kato, Masako; Murakami, Ichiro; Hayashi, Kazuhiko

2017-04-01

Graves' disease is an autoimmune disease that results in and is the most common cause of hyperthyroidism, and the reactivation of persisting Epstein-Barr virus (EBV) in B lymphocytes induces the differentiation of host B cells into plasma cells. We previously reported that some EBV-infected B cells had thyrotropin receptor antibodies (TRAbs) as surface immunoglobulins (Igs), and EBV reactivation induced these TRAb+EBV+ cells to produce TRAbs. EBV reactivation induces Ig production from host B cells. The purpose of the present study was to examine total Ig productions from B cell culture fluids and to detect activation-induced cytidine deaminase (AID), nuclear factor kappa B (NF-κB), and EBV latent membrane protein (LMP) 1 in culture B cells during EBV reactivation induction and then we discussed the mechanisms of EBV reactivation-induced Ig production in relation to autoimmunity. We showed that the EBV reactivation induces the production of every isotype of Ig and suggested that the Ig production was catalyzed by AID through LMP1 and NF-κB. The results that the amount of IgM was significantly larger compared with IgG suggested the polyclonal B cell activation due to LMP1. We proposed the pathway of EBV reactivation induced Ig production; B cells newly infected with EBV are activated by polyclonal B cell activation and produce Igs through plasma cell differentiation induced by EBV reactivation. LMP1-induced AID enabled B cells to undergo class-switch recombination to produce every isotype of Ig. According to this mechanism, EBV rescues autoreactive B cells to produce autoantibodies, which contribute to the development and exacerbation of autoimmune diseases.

Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

Energy Technology Data Exchange (ETDEWEB)

He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Li, Xiao-Dong [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Hong, Mo-Na [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Shanghai Institute of Hypertension, Shanghai (China); Chen, Qi-Zhi [Shanghai Institute of Hypertension, Shanghai (China); Han, Wei-Qing, E-mail: whan020@gmail.com [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China); Gao, Ping-Jin, E-mail: gaopingjin@sibs.ac.cn [State Key Laboratory of Medical Genetics, Shanghai Key Laboratory of Hypertension and Department of Hypertension, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (China); Laboratory of Vascular Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai (China); Shanghai Institute of Hypertension, Shanghai (China)

2016-04-29

Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.

Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

International Nuclear Information System (INIS)

He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li; Li, Xiao-Dong; Hong, Mo-Na; Chen, Qi-Zhi; Han, Wei-Qing; Gao, Ping-Jin

2016-01-01

Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediated Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.

Acupuncture inhibits cue-induced heroin craving and brain activation.

Science.gov (United States)

Cai, Xinghui; Song, Xiaoge; Li, Chuanfu; Xu, Chunsheng; Li, Xiliang; Lu, Qi

2012-11-25

Previous research using functional MRI has shown that specific brain regions associated with drug dependence and cue-elicited heroin craving are activated by environmental cues. Craving is an important trigger of heroin relapse, and acupuncture may inhibit craving. In this study, we performed functional MRI in heroin addicts and control subjects. We compared differences in brain activation between the two groups during heroin cue exposure, heroin cue exposure plus acupuncture at the Zusanli point (ST36) without twirling of the needle, and heroin cue exposure plus acupuncture at the Zusanli point with twirling of the needle. Heroin cue exposure elicited significant activation in craving-related brain regions mainly in the frontal lobes and callosal gyri. Acupuncture without twirling did not significantly affect the range of brain activation induced by heroin cue exposure, but significantly changed the extent of the activation in the heroin addicts group. Acupuncture at the Zusanli point with twirling of the needle significantly decreased both the range and extent of activation induced by heroin cue exposure compared with heroin cue exposure plus acupuncture without twirling of the needle. These experimental findings indicate that presentation of heroin cues can induce activation in craving-related brain regions, which are involved in reward, learning and memory, cognition and emotion. Acupuncture at the Zusanli point can rapidly suppress the activation of specific brain regions related to craving, supporting its potential as an intervention for drug craving.

Chronophin activation is necessary in Doxorubicin-induced actin cytoskeleton alteration.

Science.gov (United States)

Lee, Su Jin; Park, Jeen Woo; Kang, Beom Sik; Lee, Dong-Seok; Lee, Hyun-Shik; Choi, Sooyoung; Kwon, Oh-Shin

2017-06-01

Although doxorubicin (Dox)-induced oxidative stress is known to be associated with cytotoxicity, the precise mechanism remains unclear. Genotoxic stress not only generates free radicals, but also affects actin cytoskeleton stability. We showed that Dox-induced RhoA signaling stimulated actin cytoskeleton alterations, resulting in central stress fiber disruption at early time points and cell periphery cortical actin formation at a later stage, in HeLa cells. Interestingly, activation of a cofilin phosphatase, chronophin (CIN), was initially evoked by Dox-induced RhoA signaling, resulting in a rapid phosphorylated cofilin turnover leading to actin cytoskeleton remodeling. In addition, a novel interaction between CIN and 14-3-3ζ was detected in the absence of Dox treatment. We demonstrated that CIN activity is quite contrary to 14-3-3ζ binding, and the interaction leads to enhanced phosphorylated cofilin levels. Therefore, initial CIN activation regulation could be critical in Dox-induced actin cytoskeleton remodeling through RhoA/cofilin signaling. [BMB Reports 2017; 50(6): 335-340].

Clinical implications of the BRAF mutation in papillary thyroid carcinoma and chronic lymphocytic thyroiditis.

Science.gov (United States)

Kim, Woon Won; Ha, Tae Kwun; Bae, Sung Kwon

2018-01-09

The purpose of this study was to examine the possible prognostics and clinicopathologic characteristics underlying the BRAFV600E mutation and papillary thyroid carcinoma (PTC) coexisting or in absence of chronic lymphocytic thyroiditis (CLT). This study was conducted on 172 patients who had undergone total thyroidectomy or unilateral total thyroidectomy for PTC; the patients were then examined for the BRAFV600E mutation using specimens obtained after their surgery from January 2013 to August 2015. BRAF mutations were found in 130 of 172 patients (75.6%). CLT was present in 27.9% of patients (48/172). The incidence of the BRAFV600E mutation was significantly increased in the group with no CLT (P = 0.001). The findings of the multivariate analysis pertaining to the coexistence of CLT and PTC showed no significant correlation other than the BRAFV600E mutation. No significant difference was noted in the clinicopathologic factors between the two groups based on the coexistence of CLT in univariate and multivariate analyses. The BRAFV600E mutation is less frequent in PTC coexisting with CLT presumably because CLT and the BRAFV600E mutation operate independently in the formation and progression of thyroid cancer.

HTLV-1 Tax-induced NFκB activation is independent of Lys-63-linked-type polyubiquitination

International Nuclear Information System (INIS)

Gohda, Jin; Irisawa, Masato; Tanaka, Yuetsu; Sato, Shintaro; Ohtani, Kiyoshi; Fujisawa, Jun-ichi; Inoue, Jun-ichiro

2007-01-01

Human T-cell leukemia virus type 1 (HTLV-1) Tax-induced activation of nuclear factor-κB (NFκB) is thought to play a critical role in T-cell transformation and onset of adult T-cell leukemia. However, the molecular mechanism of the Tax-induced NFκB activation remains unknown. One of the mitogen-activated protein kinase kinase kinses (MAP3Ks) members, TAK1, plays a critical role in cytokine-induced activation of NFκB, which involves lysine 63-linked (K63) polyubiquitination of NEMO, a noncatalytic subunit of the IκB kinase complex. Here we show that Tax induces K63 polyubiquitination of NEMO. However, TAK1 is dispensable for Tax-induced NFκB activation, and deubiquitination of the K63 polyubiquitin chain failed to block Tax-induced NFκB activation. In addition, silencing of other MAP3Ks, including MEKK1, MEKK3, NIK, and TPL-2, did not affect Tax-induced NFκB activation. These results strongly suggest that unlike cytokine signaling, Tax-induced NFκB activation does not involve K63 polyubiquitination-mediated MAP3K activation

Fructokinase activity mediates dehydration-induced renal injury.

Science.gov (United States)

Roncal Jimenez, Carlos A; Ishimoto, Takuji; Lanaspa, Miguel A; Rivard, Christopher J; Nakagawa, Takahiko; Ejaz, A Ahsan; Cicerchi, Christina; Inaba, Shinichiro; Le, MyPhuong; Miyazaki, Makoto; Glaser, Jason; Correa-Rotter, Ricardo; González, Marvin A; Aragón, Aurora; Wesseling, Catharina; Sánchez-Lozada, Laura G; Johnson, Richard J

2014-08-01

The epidemic of chronic kidney disease in Nicaragua (Mesoamerican nephropathy) has been linked with recurrent dehydration. Here we tested whether recurrent dehydration may cause renal injury by activation of the polyol pathway, resulting in the generation of endogenous fructose in the kidney that might subsequently induce renal injury via metabolism by fructokinase. Wild-type and fructokinase-deficient mice were subjected to recurrent heat-induced dehydration. One group of each genotype was provided water throughout the day and the other group was hydrated at night, after the dehydration. Both groups received the same total hydration in 24 h. Wild-type mice that received delayed hydration developed renal injury, with elevated serum creatinine, increased urinary NGAL, proximal tubular injury, and renal inflammation and fibrosis. This was associated with activation of the polyol pathway, with increased renal cortical sorbitol and fructose levels. Fructokinase-knockout mice with delayed hydration were protected from renal injury. Thus, recurrent dehydration can induce renal injury via a fructokinase-dependent mechanism, likely from the generation of endogenous fructose via the polyol pathway. Access to sufficient water during the dehydration period can protect mice from developing renal injury. These studies provide a potential mechanism for Mesoamerican nephropathy.

Efficient molecular screening of Lynch syndrome by specific 3' promoter methylation of the MLH1 or BRAF mutation in colorectal cancer with high-frequency microsatellite instability.

Science.gov (United States)

Nakagawa, Hitoshi; Nagasaka, Takeshi; Cullings, Harry M; Notohara, Kenji; Hoshijima, Naoko; Young, Joanne; Lynch, Henry T; Tanaka, Noriaki; Matsubara, Nagahide

2009-06-01

It is sometimes difficult to diagnose Lynch syndrome by the simple but strict clinical criteria, or even by the definitive genetic testing for causative germline mutation of mismatch repair genes. Thus, some practical and efficient screening strategy to select highly possible Lynch syndrome patients is exceedingly desirable. We performed a comprehensive study to evaluate the methylation status of whole MLH1 promoter region by direct bisulfite sequencing of the entire MLH1 promoter regions on Lynch and non-Lynch colorectal cancers (CRCs). Then, we established a convenient assay to detect methylation in key CpG islands responsible for the silencing of MLH1 expression. We studied the methylation status of MLH1 as well as the CpG island methylator phenotype (CIMP) and immunohistochemical analysis of mismatch repair proteins on 16 cases of Lynch CRC and 19 cases of sporadic CRCs with high-frequency microsatellite instability (MSI-H). Sensitivity to detect Lynch syndrome by MLH1 (CCAAT) methylation was 88% and the specificity was 84%. Positive likelihood ratio (PLR) was 5.5 and negative likelihood ratio (NLR) was 0.15. Sensitivity by mutational analysis of BRAF was 100%, specificity was 84%, PLR was 6.3 and NLR was zero. By CIMP analysis; sensitivity was 88%, specificity was 79%, PLR was 4.2, and NLR was 0.16. BRAF mutation or MLH1 methylation analysis combined with MSI testing could be a good alternative to screen Lynch syndrome patients in a cost effective manner. Although the assay for CIMP status also showed acceptable sensitivity and specificity, it may not be practical because of its rather complicated assay.

Neuropharmacology of light-induced locomotor activation.

Science.gov (United States)

Amato, Davide; Pum, Martin E; Groos, Dominik; Lauber, Andrea C; Huston, Joseph P; Carey, Robert J; de Souza Silva, Maria A; Müller, Christian P

2015-08-01

Presentation of non-aversive light stimuli for several seconds was found to reliably induce locomotor activation and exploratory-like activity. Light-induced locomotor activity (LIA) can be considered a convenient simple model to study sensory-motor activation. LIA was previously shown to coincide with serotonergic and dopaminergic activation in specific cortical areas in freely moving and anesthetized animals. In the present study we explore the neuropharmacology of LIA using a receptor antagonist/agonist approach in rats. The non-selective 5-HT2-receptor antagonist ritanserin (1.5-6 mg/kg, i.p.) dose-dependently reduced LIA. Selective antagonism of either the 5-HT2A-receptor by MDL 11,939 (0.1-0.4 mg/kg, i.p.), or the 5-HT2C-receptor by SDZ SER 082 (0.125-0.5 mg/kg, i.p.), alone or in combination, had no significant influence on LIA. Also the selective 5-HT1A-receptor antagonist, WAY 100635 (0.4 mg/kg, i.p.) did not affect LIA. Neither did the preferential dopamine D2-receptor antagonist, haloperidol (0.025-0.1 mg/kg, i.p.) nor the D2/D3-receptor agonist, quinpirole (0.025-0.5 mg/kg, i.p.) affect the expression of LIA. However, blocking the glutamatergic NMDA-receptor with phencyclidine (PCP, 1.5-6 mg/kg, i.p.) dose-dependently reduced LIA. This effect was also observed with ketamine (10 mg/kg, i.p.). These findings suggest that serotonin and dopamine receptors abundantly expressed in the cortex do not mediate light-stimulus triggered locomotor activity. PCP and ketamine effects, however, suggest an important role of NMDA receptors in LIA. Copyright © 2015 Elsevier Ltd. All rights reserved.

Autophagy activation, not peroxisome proliferator-activated receptor γ coactivator 1α, may mediate exercise-induced improvements in glucose handling during diet-induced obesity.

Science.gov (United States)

Rosa-Caldwell, Megan E; Brown, Jacob L; Lee, David E; Blackwell, Thomas A; Turner, Kyle W; Brown, Lemuel A; Perry, Richard A; Haynie, Wesley S; Washington, Tyrone A; Greene, Nicholas P

2017-09-01

What is the central question of this study? What are the individual and combined effects of muscle-specific peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) overexpression and physical activity during high-fat feeding on glucose and exercise tolerance? What is the main finding and its importance? Our main finding is that muscle-specific PGC-1α overexpression provides no protection against lipid-overload pathologies nor does it enhance exercise adaptations. Instead, physical activity, regardless of PGC-1α content, protects against high-fat diet-induced detriments. Activation of muscle autophagy was correlated with exercise protection, suggesting that autophagy might be a mediating factor for exercise-induced protection from lipid overload. The prevalence of glucose intolerance is alarmingly high. Efforts to promote mitochondrial biogenesis through peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) to mitigate glucose intolerance have been controversial. However, physical activity remains a primary means to alleviate the condition. The aim of this study was to determine the combined effects of muscle-specific overexpression of PGC-1α and physical activity on glucose handling during diet-induced obesity. Wild-type (WT, ∼20) and PGC-1α muscle transgenic (MCK-PGC-1α, ∼20) mice were given a Western diet (WD) at 8 weeks age and allowed to consume food ab libitum throughout the study. At 12 weeks of age, all animals were divided into sedentary (SED) or voluntary wheel running (VWR) interventions. At 7, 11 and 15 weeks of age, animals underwent glucose tolerance tests (GTT) and graded exercise tests (GXT). At 16 weeks of age, tissues were collected. At 11 weeks, the MCK-PGC-1α animals had 50% greater glucose tolerance integrated area under the curve compared with WT. However, at 15 weeks, SED animals also had greater GTT integrated area under the curve compared with VWR, regardless of genotype; furthermore, SED

Activation of peroxisome proliferator-activated receptor-γ (PPARγ) induces cell death through MAPK-dependent mechanism in osteoblastic cells

International Nuclear Information System (INIS)

Kim, Sung Hun; Yoo, Chong Il; Kim, Hui Taek; Park, Ji Yeon; Kwon, Chae Hwa; Keun Kim, Yong

2006-01-01

The present study was undertaken to determine the role of the mitogen-activated protein kinase (MAPK) subfamilies in cell death induced by PPARγ agonists in osteoblastic cells. Ciglitazone and troglitazone, PPARγ agonists, resulted in a concentration- and time-dependent cell death, which was largely attributed to apoptosis. But a PPARα agonist ciprofibrate did not affect the cell death. Ciglitazone caused reactive oxygen species (ROS) generation and ciglitazone-induced cell death was prevented by antioxidants, suggesting an important role of ROS generation in the ciglitazone-induced cell death. ROS generation and cell death induced by ciglitazone were inhibited by the PPARγ antagonist GW9662. Ciglitazone treatment caused activation of extracellular signal-regulated kinase (ERK) and p38. Activation of ERK was dependent on epidermal growth factor receptor (EGFR) and that of p38 was independent. Ciglitazone-induced cell death was significantly prevented by PD98059, an inhibitor of ERK upstream kinase MEK1/2, and SB203580, a p38 inhibitor. Ciglitazone treatment increased Bax expression and caused a loss of mitochondrial membrane potential, and its effect was prevented by N-acetylcysteine, PD98059, and SB203580. Ciglitazone induced caspase activation, which was prevented by PD98059 and SB203580. The general caspase inhibitor z-DEVD-FMK and the specific inhibitor of caspases-3 DEVD-CHO exerted the protective effect against the ciglitazone-induced cell death. The EGFR inhibitors AG1478 and suramin protected against the ciglitazone-induced cell death. Taken together, these findings suggest that the MAPK signaling pathways play an active role in mediating the ciglitazone-induced cell death of osteoblasts and function upstream of a mitochondria-dependent mechanism. These data may provide a novel insight into potential therapeutic strategies for treatment of osteoporosis

Kupffer cells activation promoted binge drinking-induced fatty liver by activating lipolysis in white adipose tissues.

Science.gov (United States)

Zhao, Yu-Ying; Yang, Rui; Xiao, Mo; Guan, Min-Jie; Zhao, Ning; Zeng, Tao

2017-09-01

Kupffer cells (KCs) have been suggested to play critical roles in chronic ethanol induced early liver injury, but the role of KCs in binge drinking-induced hepatic steatosis remains unclear. This study was designed to investigate the roles of KCs inhibitor (GdCl 3 ) and TNF-α antagonist (etanercept) on binge drinking-induced liver steatosis and to explore the underlying mechanisms. C57BL/6 mice were exposed to three doses of ethanol (6g/kg body weight) to mimic binge drinking-induced fatty liver. The results showed that both GdCl 3 and etanercept partially but significantly alleviated binge drinking-induced increase of hepatic triglyceride (TG) level, and reduced fat droplets accumulation in mice liver. GdCl 3 but not etanercept significantly blocked binge drinking-induced activation of KCs. However, neither GdCl 3 nor etanercept could affect binge drinking-induced decrease of PPAR-α, ACOX, FAS, ACC and SCD protein levels, or increase of the LC3 II/LC3 I ratio and p62 protein level. Interestingly, both GdCl 3 and etanercept significantly suppressed binge drinking-induced phosphorylation of HSL in epididymal adipose tissues. Results of in vitro studies with cultured epididymal adipose tissues showed that TNF-α could increase the phosphorylation of HSL in adipose tissues and upgrade the secretion of free fatty acid (FFA) in the culture medium. Taken together, KCs inhibitor and TNF-α antagonist could partially attenuate binge drinking-induced liver steatosis, which might be attributed to the suppression of mobilization of white adipose tissues. These results suggest that KCs activation may promote binge drinking-induced fatty liver by TNF-α mediated activation of lipolysis in white adipose tissues. Copyright © 2017 Elsevier B.V. All rights reserved.

Human retinal pigment epithelial cell-induced apoptosis in activated T cells

DEFF Research Database (Denmark)

Jørgensen, A; Wiencke, A K; la Cour, M

1998-01-01

human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

Nrf2 activation prevents cadmium-induced acute liver injury

International Nuclear Information System (INIS)

Wu, Kai C.; Liu, Jie J.; Klaassen, Curtis D.

2012-01-01

Oxidative stress plays an important role in cadmium-induced liver injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that up-regulates cytoprotective genes in response to oxidative stress. To investigate the role of Nrf2 in cadmium-induced hepatotoxicity, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation were treated with cadmium chloride (3.5 mg Cd/kg, i.p.). Blood and liver samples were collected 8 h thereafter. Cadmium increased serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities, and caused extensive hepatic hemorrhage and necrosis in the Nrf2-null mice. In contrast, Nrf2-enhanced mice had lower serum ALT and LDH activities and less morphological alternations in the livers than wild-type mice. H 2 DCFDA (2′,7′-dichlorodihydrofluoresein diacetate) staining of primary hepatocytes isolated from the four genotypes of mice indicated that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. To further investigate the mechanism of the protective effect of Nrf2, mRNA of metallothionein (MT) and other cytoprotective genes were determined. Cadmium markedly induced MT-1 and MT-2 in livers of all four genotypes of mice. In contrast, genes involved in glutathione synthesis and reducing reactive oxygen species, including glutamate-cysteine ligase (Gclc), glutathione peroxidase-2 (Gpx2), and sulfiredoxin-1 (Srxn-1) were only induced in Nrf2-enhanced mice, but not in Nrf2-null mice. In conclusion, the present study shows that Nrf2 activation prevents cadmium-induced oxidative stress and liver injury through induction of genes involved in antioxidant defense rather than genes that scavenge Cd. -- Highlights: ► Cadmium caused extensive hepatic hemorrhage and necrosis in Nrf2-null mice. ► Keap1-KD and Keap1-HKO mice were

Nrf2 activation prevents cadmium-induced acute liver injury

Energy Technology Data Exchange (ETDEWEB)

Wu, Kai C. [Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Liu, Jie J. [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Klaassen, Curtis D., E-mail: cklaasse@kumc.edu [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States)

2012-08-15

Oxidative stress plays an important role in cadmium-induced liver injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that up-regulates cytoprotective genes in response to oxidative stress. To investigate the role of Nrf2 in cadmium-induced hepatotoxicity, Nrf2-null mice, wild-type mice, kelch-like ECH-associated protein 1-knockdown (Keap1-KD) mice with enhanced Nrf2, and Keap1-hepatocyte knockout (Keap1-HKO) mice with maximum Nrf2 activation were treated with cadmium chloride (3.5 mg Cd/kg, i.p.). Blood and liver samples were collected 8 h thereafter. Cadmium increased serum alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities, and caused extensive hepatic hemorrhage and necrosis in the Nrf2-null mice. In contrast, Nrf2-enhanced mice had lower serum ALT and LDH activities and less morphological alternations in the livers than wild-type mice. H{sub 2}DCFDA (2′,7′-dichlorodihydrofluoresein diacetate) staining of primary hepatocytes isolated from the four genotypes of mice indicated that oxidative stress was higher in Nrf2-null cells, and lower in Nrf2-enhanced cells than in wild-type cells. To further investigate the mechanism of the protective effect of Nrf2, mRNA of metallothionein (MT) and other cytoprotective genes were determined. Cadmium markedly induced MT-1 and MT-2 in livers of all four genotypes of mice. In contrast, genes involved in glutathione synthesis and reducing reactive oxygen species, including glutamate-cysteine ligase (Gclc), glutathione peroxidase-2 (Gpx2), and sulfiredoxin-1 (Srxn-1) were only induced in Nrf2-enhanced mice, but not in Nrf2-null mice. In conclusion, the present study shows that Nrf2 activation prevents cadmium-induced oxidative stress and liver injury through induction of genes involved in antioxidant defense rather than genes that scavenge Cd. -- Highlights: ► Cadmium caused extensive hepatic hemorrhage and necrosis in Nrf2-null mice. ► Keap1-KD and Keap1-HKO mice

Laser Induced Selective Activation For Subsequent Autocatalytic Electroless Plating

DEFF Research Database (Denmark)

Zhang, Yang

. The third hypothesis is that the activation and rinsing process can be described by diffusion. This hypothesis is proved using Fick’s diffusion laws combined with the short-time-plating experiment. The influence of laser parameters on the surface structure is investigated for Nd:YAG, UV, and fiber lasers......The subject of this PhD thesis is “Laser induced selective activation for subsequent autocatalytic electroless plating.” The objective of the project is to investigate the process chains for micro structuring of polymer surfaces for selective micro metallization. Laser induced selective activation...... (LISA) is introduced and studied as a new technique for producing 3D moulded interconnect devices (3D-MIDs). This technique enables the metallization of polymer surface modified by laser and subsequently activated by a PdCl2/SnCl2 system. Various technologies exist on an industrial level...

Kefiran suppresses antigen-induced mast cell activation.

Science.gov (United States)

Furuno, Tadahide; Nakanishi, Mamoru

2012-01-01

Kefir is a traditional fermented milk beverage produced by kefir grains in the Caucasian countries. Kefiran produced by Lactobacillus kefiranofaciens in kefir grains is an exopolysaccharide having a repeating structure with glucose and galactose residues in the chain sequence and has been suggested to exert many health-promoting effects such as immunomodulatory, hypotensive, hypocholesterolemic activities. Here we investigated the effects of kefiran on mast cell activation induced by antigen. Pretreatment with kefiran significantly inhibited antigen-induced Ca(2+) mobilization, degranulation, and tumor necrosis factor-α production in bone marrow-derived mast cells (BMMCs) in a dose-dependent manner. The phosphorylation of Akt, glycogen synthase kinase 3β, and extracellular signal-regulated kinases (ERKs) after antigen stimulation was also suppressed by pretreatment of BMMCs with kefiran. These findings indicate that kefiran suppresses mast cell degranulation and cytokine production by inhibiting the Akt and ERKs pathways, suggesting an anti-inflammatory effect for kefiran.

Minocycline attenuates sevoflurane-induced cell injury via activation of Nrf2

Science.gov (United States)

Tian, Yue; Wu, Xiuying; Guo, Shanbin; Ma, Ling; Huang, Wei; Zhao, Xiaochun

2017-01-01

Minocycline has been demonstrated to exert neuroprotective effects in various experimental models. In the present study, we investigated the mechanisms underlying the protective effects of minocycline on cell injury induced by the inhalation of the anesthetic, sevoflurane. In our in vivo experiments using rats, minocycline attenuated sevoflurane-induced neuronal degeneration and apoptosis in the rat hippocampus, and this effect was associated with the minocycline-mediated suppression of oxidative stress in the hippocampus. In in vitro experiments, minocycline inhibited sevoflurane-induced apoptosis and the production of reactive oxygen species (ROS) in H4 human neuroglioma cells. In addition, minocycline suppressed the sevoflurane-induced upregulation of interleukin (IL)-6 and the activation of the nuclear factor-κB (NF-κB) signaling pathway in H4 cells. Furthermore, we found that nuclear factor E2-related factor 2 (Nrf2), an activator of the stress response, was upregulated and activated upon sevoflurane treatment both in the rat hippocampus and in H4 cells. In addition, minocycline further augmented the upregulation and activation of Nrf2 when used in conjunction with sevoflurane. Moreover, the knockdown of Nrf2 in H4 cells by small interfering RNA (siRNA) diminished the cytoprotective effect of minocycline, and attenuated the inhibitory effect of minocycline on ROS production, IL-6 upregulation and the activation of the NF-κB signaling pathway. On the whole, our findings indicate that minocycline may exert protective effects against sevoflurane-induced cell injury via the Nrf2-modulated antioxidant response and the inhibition of the activation of the NF-κB signaling pathway. PMID:28260081

mTOR inhibition sensitizes ONC201-induced anti-colorectal cancer cell activity.

Science.gov (United States)

Jin, Zhe-Zhu; Wang, Wei; Fang, Di-Long; Jin, Yong-Jun

2016-09-30

We here tested the anti-colorectal cancer (CRC) activity by a first-in-class small molecule TRAIL inducer ONC201. The potential effect of mTOR on ONC201's actions was also examined. ONC201 induced moderate cytotoxicity against CRC cell lines (HT-29, HCT-116 and DLD-1) and primary human CRC cells. Significantly, AZD-8055, a mTOR kinase inhibitor, sensitized ONC201-induced cytotoxicity in CRC cells. Meanwhile, ONC201-induced TRAIL/death receptor-5 (DR-5) expression, caspase-8 activation and CRC cell apoptosis were also potentiated with AZD-8055 co-treatment. Reversely, TRAIL sequestering antibody RIK-2 or the caspase-8 specific inhibitor z-IETD-fmk attenuated AZD-8055 plus ONC201-induced CRC cell death. Further, mTOR kinase-dead mutation (Asp-2338-Ala) or shRNA knockdown significantly sensitized ONC201's activity in CRC cells, leading to profound cell death and apoptosis. On the other hand, expression of a constitutively-active S6K1 (T389E) attenuated ONC201-induced CRC cell apoptosis. For the mechanism study, we showed that ONC201 blocked Akt, but only slightly inhibited mTOR in CRC cells. Co-treatment with AZD-8055 also concurrently blocked mTOR activation. These results suggest that mTOR could be a primary resistance factor of ONC201 in CRC cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Effects of Active Mastication on Chronic Stress-Induced Bone Loss in Mice.

Science.gov (United States)

Azuma, Kagaku; Furuzawa, Manabu; Fujiwara, Shu; Yamada, Kumiko; Kubo, Kin-ya

2015-01-01

Chronic psychologic stress increases corticosterone levels, which decreases bone density. Active mastication or chewing attenuates stress-induced increases in corticosterone. We evaluated whether active mastication attenuates chronic stress-induced bone loss in mice. Male C57BL/6 (B6) mice were randomly divided into control, stress, and stress/chewing groups. Stress was induced by placing mice in a ventilated restraint tube (60 min, 2x/day, 4 weeks). The stress/chewing group was given a wooden stick to chew during the experimental period. Quantitative micro-computed tomography, histologic analysis, and biochemical markers were used to evaluate the bone response. The stress/chewing group exhibited significantly attenuated stress-induced increases in serum corticosterone levels, suppressed bone formation, enhanced bone resorption, and decreased trabecular bone mass in the vertebrae and distal femurs, compared with mice in the stress group. Active mastication during exposure to chronic stress alleviated chronic stress-induced bone density loss in B6 mice. Active mastication during chronic psychologic stress may thus be an effective strategy to prevent and/or treat chronic stress-related osteopenia.

Clinical significance of plasminogen activator inhibitor activity in patients with exercise-induced ischemia

International Nuclear Information System (INIS)

Sakata, K.; Kurata, C.; Taguchi, T.; Suzuki, S.; Kobayashi, A.; Yamazaki, N.; Rydzewski, A.; Takada, Y.; Takada, A.

1990-01-01

To assess the fibrinolytic system in patients with exercise-induced ischemia and its relation to ischemia and severity of coronary artery disease (CAD), 47 patients with CAD confirmed by results of coronary angiography underwent symptom-limited multistage exercise thallium-201 emission computed tomography. All patients with CAD had exercise-induced ischemia as assessed from thallium-201 images. Pre- and peak exercise blood samples from each patient and preexercise blood samples from control subjects were assayed for several fibrinolytic components and were also assayed for plasma adrenaline. The extent of ischemia was defined as delta visual uptake score (total visual uptake score in delayed images minus total visual uptake score in initial images) and the severity of CAD as the number of diseased vessels. In the basal condition, plasminogen activator inhibitor (PAI) activity was significantly higher in patients with exercise-induced ischemia as compared to control subjects (p less than 0.01), although there were no significant differences in other fibrinolytic variables between the two groups. Moreover, PAI activity in the basal condition displayed a significantly positive correlation with the extent of ischemia (r = 0.47, p less than 0.01). Patients with exercise-induced ischemia were divided into two groups (24 with single-vessel disease and 23 with multivessel disease). There were no significant differences in coronary risk factors, hemodynamics, or plasma adrenaline levels during exercise between single-vessel and multivessel disease except that delta visual uptake score was significantly higher in multivessel disease (p less than 0.01)

Temporal lobe pleomorphic xanthoastrocytoma and acquired BRAF mutation in an adolescent with the constitutional 22q11.2 deletion syndrome.

Science.gov (United States)

Murray, Jeffrey C; Donahue, David J; Malik, Saleem I; Dzurik, Yvette B; Braly, Emily Z; Dougherty, Margaret J; Eaton, Katherine W; Biegel, Jaclyn A

2011-05-01

DiGeorge syndrome, or velocardiofacial syndrome (DGS/VCFS), is a rare and usually sporadic congenital genetic disorder resulting from a constitutional microdeletion at chromosome 22q11.2. While rare cases of malignancy have been described, likely due to underlying immunodeficiency, central nervous system tumors have not yet been reported. We describe an adolescent boy with DGS/VCFS who developed a temporal lobe pleomorphic xanthoastrocytoma. High-resolution single nucleotide polymorphism array studies of the tumor confirmed a constitutional 22q11.21 deletion, and revealed acquired gains, losses and copy number neutral loss of heterozygosity of several chromosomal regions, including a homozygous deletion of the CDKN2A/B locus. The tumor also demonstrated a common V600E mutation in the BRAF oncogene. This is the first reported case of a patient with DiGeorge syndrome developing a CNS tumor of any histology and expands our knowledge about low-grade CNS tumor molecular genetics.

Resting-state brain activity in the motor cortex reflects task-induced activity: A multi-voxel pattern analysis.

Science.gov (United States)

Kusano, Toshiki; Kurashige, Hiroki; Nambu, Isao; Moriguchi, Yoshiya; Hanakawa, Takashi; Wada, Yasuhiro; Osu, Rieko

2015-08-01

It has been suggested that resting-state brain activity reflects task-induced brain activity patterns. In this study, we examined whether neural representations of specific movements can be observed in the resting-state brain activity patterns of motor areas. First, we defined two regions of interest (ROIs) to examine brain activity associated with two different behavioral tasks. Using multi-voxel pattern analysis with regularized logistic regression, we designed a decoder to detect voxel-level neural representations corresponding to the tasks in each ROI. Next, we applied the decoder to resting-state brain activity. We found that the decoder discriminated resting-state neural activity with accuracy comparable to that associated with task-induced neural activity. The distribution of learned weighted parameters for each ROI was similar for resting-state and task-induced activities. Large weighted parameters were mainly located on conjunctive areas. Moreover, the accuracy of detection was higher than that for a decoder whose weights were randomly shuffled, indicating that the resting-state brain activity includes multi-voxel patterns similar to the neural representation for the tasks. Therefore, these results suggest that the neural representation of resting-state brain activity is more finely organized and more complex than conventionally considered.

Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus

International Nuclear Information System (INIS)

Banerjee, Chaitali; Goswami, Ramansu; Datta, Soma; Rajagopal, R.; Mazumder, Shibnath

2011-01-01

We had earlier shown that exposure to arsenic (0.50 μM) caused caspase-3 mediated head kidney macrophage (HKM) apoptosis involving the p38-JNK pathway in Clarias batrachus. Here we examined the roles of calcium (Ca 2+ ) and extra-cellular signal-regulated protein kinase (ERK), the other member of MAPK-pathway on arsenic-induced HKM apoptosis. Arsenic-induced HKM apoptosis involved increased expression of ERK and calpain-2. Nifedipine, verapamil and EGTA pre-treatment inhibited the activation of calpain-2, ERK and reduced arsenic-induced HKM apoptosis as evidenced from reduced caspase-3 activity, Annexin V-FITC-propidium iodide and Hoechst 33342 staining. Pre-incubation with ERK inhibitor U 0126 inhibited the activation of calpain-2 and interfered with arsenic-induced HKM apoptosis. Additionally, pre-incubation with calpain-2 inhibitor also interfered with the activation of ERK and inhibited arsenic-induced HKM apoptosis. The NADPH oxidase inhibitor apocynin and diphenyleneiodonium chloride also inhibited ERK activation indicating activation of ERK in arsenic-exposed HKM also depends on signals from NADPH oxidase pathway. Our study demonstrates the critical role of Ca 2+ homeostasis on arsenic-induced HKM apoptosis. We suggest that arsenic-induced alteration in intracellular Ca 2+ levels initiates pro-apoptotic ERK and calpain-2; the two pathways influence each other positively and induce caspase-3 mediated HKM apoptosis. Besides, our study also indicates the role of ROS in the activation of ERK pathway in arsenic-induced HKM apoptosis in C. batrachus. - Highlights: → Altered Ca 2+ homeostasis leads to arsenic-induced HKM apoptosis. → Calpain-2 plays a critical role in the process. → ERK is pro-apoptotic in arsenic-induced HKM apoptosis. → Arsenic-induced HKM apoptosis involves cross talk between calpain-2 and ERK.

Activation of PPARγ is not involved in butyrate-induced epithelial cell differentiation

International Nuclear Information System (INIS)

Ulrich, S.; Waechtershaeuser, A.; Loitsch, S.; Knethen, A. von; Bruene, B.; Stein, J.

2005-01-01

Histone deacetylase-inhibitors affect growth and differentiation of intestinal epithelial cells by inducing expression of several transcription factors, e.g. Peroxisome proliferator-activated receptor γ (PPARγ) or vitamin D receptor (VDR). While activation of VDR by butyrate mainly seems to be responsible for cellular differentiation, the activation of PPARγ in intestinal cells remains to be elucidated. The aim of this study was to determine the role of PPARγ in butyrate-induced cell growth inhibition and differentiation induction in Caco-2 cells. Treatment with PPARγ ligands ciglitazone and BADGE (bisphenol A diglycidyl) enhanced butyrate-induced cell growth inhibition in a dose- and time-dependent manner, whereas cell differentiation was unaffected after treatment with PPARγ ligands rosiglitazone and MCC-555. Experiments were further performed in dominant-negative PPARγ mutant cells leading to an increase in cell growth whereas butyrate-induced cell differentiation was again unaffected. The present study clearly demonstrated that PPARγ is involved in butyrate-induced inhibition of cell growth, but seems not to play an essential role in butyrate-induced cell differentiation

Regulation of radiation-induced protein kinase Cδ activation in radiation-induced apoptosis differs between radiosensitive and radioresistant mouse thymic lymphoma cell lines

International Nuclear Information System (INIS)

Nakajima, Tetsuo; Yukawa, Osami; Tsuji, Hideo; Ohyama, Harumi; Wang, Bing; Tatsumi, Kouichi; Hayata, Isamu; Hama-Inaba, Hiroko

2006-01-01

Protein kinase Cδ (PKCδ) has an important role in radiation-induced apoptosis. The expression and function of PKCδ in radiation-induced apoptosis were assessed in a radiation-sensitive mouse thymic lymphoma cell line, 3SBH5, and its radioresistant variant, XR223. Rottlerin, a PKCδ-specific inhibitor, completely abolished radiation-induced apoptosis in 3SBH5. Radiation-induced PKCδ activation correlated with the degradation of PKCδ, indicating that PKCδ activation through degradation is involved in radiation-induced apoptosis in radiosensitive 3SBH5. In radioresistant XR223, radiation-induced PKCδ activation was lower than that in radiosensitive 3SBH5. Cytosol PKCδ levels in 3SBH5 decreased markedly after irradiation, while those in XR223 did not. There was no apparent change after irradiation in the membrane fractions of either cell type. In addition, basal cytosol PKCδ levels in XR223 were higher than those in 3SBH5. These results suggest that the radioresistance in XR223 to radiation-induced apoptosis is due to a difference in the regulation of radiation-induced PKCδ activation compared to that of 3SBH5. On the other hand, Atm -/- mouse thymic lymphoma cells were more radioresistant to radiation-induced apoptosis than wild-type mouse thymic lymphoma cells. Irradiated wild-type cells, but not Atm -/- cells, had decreased PKCδ levels, indicating that the Atm protein is involved in radiation-induced apoptosis through the induction of PKCδ degradation. The decreased Atm protein levels induced by treatment with Atm small interfering RNA had no effect on radiation-induced apoptosis in 3SBH5 cells. These results suggest that the regulation of radiation-induced PKCδ activation, which is distinct from the Atm-mediated cascade, determines radiation sensitivity in radiosensitive 3SBH5 cells

The phosphorylation specificity of B-RAF(WT), B-RAF(D594V), B-RAF(V600E) and B-RAF(K601E) kinases: An in silico study

DEFF Research Database (Denmark)

Fratev, Filip Filipov; Jonsdottir, Svava Osk

2010-01-01

Phosphorylation of the B-RAF kinase is an essential process in tumour induction and maintenance in several cancers. Herein the phosphorylation specificity of the activation segment of the wild type B-RAF kinase and the B-RAF(D594V), B-RAF(V600E) and B-RAF(K601E) mutants was examined by molecular ...

Characteristics of induced activity from medical linear accelerators

International Nuclear Information System (INIS)

Wang Yizhen; Evans, Michael D.C.; Podgorsak, Ervin B.

2005-01-01

A study of the induced activity in a medical linear accelerator (linac) room was carried out on several linac installations. Higher beam energy, higher dose rate, and larger field size generally result in higher activation levels at a given point of interest, while the use of multileaf collimators (MLC) can also increase the activation level at the isocenter. Both theoretical and experimental studies reveal that the activation level in the morning before any clinical work increases from Monday to Saturday and then decreases during the weekend. This weekly activation picture keeps stable from one week to another during standard clinical operation of the linac. An effective half-life for a given point in the treatment room can be determined from the measured or calculated activity decay curves. The effective half-life for points inside the treatment field is longer than that for points outside of the field in the patient plane, while a larger field and longer irradiation time can also make the effective half-life longer. The activation level reaches its practical saturation value after a 30 min continuous irradiation, corresponding to 12 000 MU at a 'dose rate' of 400 MU/min. A 'dose' of 300 MU was given 20 times in 15 min intervals to determine the trends in the activation level in a typical clinical mode. As well, a long-term (85 h over a long weekend) decay curve was measured to evaluate the long-term decay of room activation after a typical day of clinical linac use. A mathematical model for the activation level at the isocenter has been established and shown to be useful in explaining and predicting the induced activity levels for typical clinical and experimental conditions. The activation level for a 22 MeV electron beam was also measured and the result shows it is essentially negligible

[Clinical and economic evaluation of the introduction of the combinazion trametinib + dabrafenib in the management of advanced melanoma in the Italian market

Directory of Open Access Journals (Sweden)

Lorenzo Pradelli

2016-12-01

Full Text Available Melanoma is the most aggressive type of all skin cancers. In Italy the incidence is increasing both in men and in women with 13,800 new cases expected in 2016. The advanced melanoma therapy has changed in recent years with the use of immunotherapy and targeted therapies. In particular, treatment with BRAF inhibitors in patients with advanced BRAF V600 mutated melanoma has shown high rates of rapid response and survival. Due to development of acquired resistance with disease progression the rapid response observed with BRAF inhibitor therapy is not long lasting. Combining a BRAF inhibitor with a MEK inhibitor may help to delay the development of resistance and to enhance the antitumor activities with a further increase in the response and survival rate. Trametinib, an inhibitor of MEK kinases, and dabrafenib, an inhibitor of BRAF kinase, have authorizations as monotherapies and in combination with each other for treating adults with unresectable or metastatic melanoma with BRAF V600 mutation. Purpose of this report is to describe the combination in terms of clinical efficacy, safety, and economic impact. In particular, a cost-effectiveness analysis and a budget impact analysis were performed in order to evaluate the combination versus monotherapy and the financial sustainability of trametinib + dabrafenib on the Italian market. [In Italian

Vemurafenib for the treatment of melanoma.

LENUS (Irish Health Repository)

Jordan, Emmet John

2012-12-01

Metastatic melanoma is an aggressive disease resistant to chemotherapy. Recent clinical trials have reported improved survival for two novel agents; ipilimumab, a humanized, IgG1 monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and vemurafenib , a BRAF (v-raf murine sarcoma viral oncogene homolog B1) inhibitor targeting an activating mutation in the serine-threonine-protein kinase BRAF gene. AREAS COVERED: The authors reviewed preclinical and clinical data examining the safety of vemurafenib in melanoma. MEDLINE and EMBASE were searched using the medical subject heading \\'vemurafenib\\' and the following text terms: melanoma, BRAF inhibition, vemurafenib. This review provides the reader with an overview of current data examining the efficacy and safety of vemurafenib in metastatic melanoma. EXPERT OPINION: Vemurafenib is an oral agent licensed for patients with BRAF V600E mutation-positive inoperable and metastatic melanoma. The most common adverse effects observed in Phase III clinical trials were dermatological events, arthralgia and fatigue. Specific dermatological toxicities included development of cutaneous squamous cell cancers and keratoacanthomas. Prolongation of the QT interval was also reported. Regular dermatological assessments and electrocardiograms are recommended. Ongoing trials are examining vemurafenib in both the adjuvant setting and metastatic setting in combination with ipilimumab and MEK inhibitors (mitogen-activated protein kinase\\/extracellular signal-regulated kinase). Understanding and overcoming mechanisms of resistance to BRAF inhibitors is the focus of ongoing research.

Chemically induced and light-independent cryptochrome photoreceptor activation.

Science.gov (United States)

Rosenfeldt, Gesa; Viana, Rafael Muñoz; Mootz, Henning D; von Arnim, Albrecht G; Batschauer, Alfred

2008-01-01

The cryptochrome photoreceptors of higher plants are dimeric proteins. Their N-terminal photosensory domain mediates dimerization, and the unique C-terminal extension (CCT) mediates signaling. We made use of the human FK506-binding protein (FKBP) that binds with high affinity to rapamycin or rapamycin analogs (rapalogs). The FKBP-rapamycin complex is recognized by another protein, FRB, thus allowing rapamycin-induced dimerization of two target proteins. Here we demonstrate by bioluminescence resonance energy transfer (BRET) assays the applicability of this regulated dimerization system to plants. Furthermore, we show that fusion proteins consisting of the C-terminal domain of Arabidopsis cryptochrome 2 fused to FKBP and FRB and coexpressed in Arabidopsis cells specifically induce the expression of cryptochrome-controlled reporter and endogenous genes in darkness upon incubation with the rapalog. These results demonstrate that the activation of cryptochrome signal transduction can be chemically induced in a dose-dependent fashion and uncoupled from the light signal, and provide the groundwork for gain-of-function experiments to study specifically the role of photoreceptors in darkness or in signaling cross-talk even under light conditions that activate members of all photoreceptor families.

A synthetic peptide blocking TRPV1 activation inhibits UV-induced skin responses.

Science.gov (United States)

Kang, So Min; Han, Sangbum; Oh, Jang-Hee; Lee, Young Mee; Park, Chi-Hyun; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

2017-10-01

Transient receptor potential type 1 (TRPV1) can be activated by ultraviolet (UV) irradiation, and mediates UV-induced matrix metalloproteinase (MMP)-1 and proinflammatory cytokines in keratinocytes. Various chemicals and compounds targeting TRPV1 activation have been developed, but are not in clinical use mostly due to their safety issues. We aimed to develop a novel TRPV1-targeting peptide to inhibit UV-induced responses in human skin. We designed and generated a novel TRPV1 inhibitory peptide (TIP) which mimics the specific site in TRPV1 (aa 701-709: Gln-Arg-Ala-Ile-Thr-Ile-Leu-Asp-Thr, QRAITILDT), Thr 705 , and tested its efficacy of blocking UV-induced responses in HaCaT, mouse, and human skin. TIP effectively inhibited capsaicin-induced calcium influx and TRPV1 activation. Treatment of HaCaT with TIP prevented UV-induced increases of MMP-1 and pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor-α. In mouse skin in vivo, TIP inhibited UV-induced skin thickening and prevented UV-induced expression of MMP-13 and MMP-9. Moreover, TIP attenuated UV-induced erythema and the expression of MMP-1, MMP-2, IL-6, and IL-8 in human skin in vivo. The novel synthetic peptide targeting TRPV1 can ameliorate UV-induced skin responses in vitro and in vivo, providing a promising therapeutic approach against UV-induced inflammation and photoaging. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Induced resistance in tomato by SAR activators during predisposing salinity stress

Directory of Open Access Journals (Sweden)

Matthew Francis Pye

2013-05-01

Full Text Available Plant activators are chemicals that induce disease resistance. The phytohormone salicylic acid (SA is a crucial signal for systemic acquired resistance (SAR, and SA-mediated resistance is a target of several commercial plant activators, including Actigard (1,2,3-benzothiadiazole-7-thiocarboxylic acid-s-methyl-ester, BTH and Tiadinil (N-(3-chloro-4-methylphenyl-4-methyl-1,2,3-thiadiazole-5-carboxamide, TDL. BTH and TDL were examined for their impact on abscisic acid (ABA-mediated, salt-induced disease predisposition in tomato seedlings. A brief episode of salt stress to roots significantly increased the severity of disease caused by Pseudomonas syringae pv. tomato (Pst and Phytophthora capsici relative to non-stressed plants. Root treatment with TDL induced resistance to Pst in leaves and provided protection in both non-stressed and salt-stressed seedlings in WT and highly susceptible NahG plants. Non-stressed and salt-stressed ABA-deficient sitiens mutants were highly resistant to Pst. Neither TDL nor BTH induced resistance to root infection by P. capsici, nor did they moderate the salt-induced increment in disease severity. Root treatment with these plant activators increased the levels of ABA in roots and shoots similar to levels observed in salt-stressed plants. The results indicate that SAR activators can protect tomato plants from bacterial speck disease under predisposing salt stress, and suggest that some SA-mediated defense responses function sufficiently in plants with elevated levels of ABA.

Activation of Rho GTPases by Cytotoxic Necrotizing Factor 1 Induces Macropinocytosis and Scavenging Activity in Epithelial Cells

Science.gov (United States)

Fiorentini, Carla; Falzano, Loredana; Fabbri, Alessia; Stringaro, Annarita; Logozzi, Mariaantonia; Travaglione, Sara; Contamin, Stéphanette; Arancia, Giuseppe; Malorni, Walter; Fais, Stefano

2001-01-01

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced “switching on” of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages. PMID:11452003

Lytic cell death induced by melittin bypasses pyroptosis but induces NLRP3 inflammasome activation and IL-1β release.

Science.gov (United States)

Martín-Sánchez, Fátima; Martínez-García, Juan José; Muñoz-García, María; Martínez-Villanueva, Miriam; Noguera-Velasco, José A; Andreu, David; Rivas, Luís; Pelegrín, Pablo

2017-08-10

The nucleotide-binding domain and leucine-rich repeat-containing receptor with a pyrin domain 3 (NLRP3) inflammasome is a sensor for different types of infections and alterations of homeostatic parameters, including abnormally high levels of the extracellular nucleotide ATP or crystallization of different metabolites. All NLRP3 activators trigger a similar intracellular pathway, where a decrease in intracellular K + concentration and permeabilization of plasma membrane are key steps. Cationic amphipathic antimicrobial peptides and peptide toxins permeabilize the plasma membrane. In fact, some of them have been described to activate the NLRP3 inflammasome. Among them, the bee venom antimicrobial toxin peptide melittin is known to elicit an inflammatory reaction via the NLRP3 inflammasome in response to bee venom. Our study found that melittin induces canonical NLRP3 inflammasome activation by plasma membrane permeabilization and a reduction in the intracellular K + concentration. Following melittin treatment, the apoptosis-associated speck-like protein, an adaptor protein with a caspase recruitment domain (ASC), was necessary to activate caspase-1 and induce IL-1β release. However, cell death induced by melittin prevented the formation of large ASC aggregates, amplification of caspase-1 activation, IL-18 release and execution of pyroptosis. Therefore, melittin-induced activation of the NLRP3 inflammasome results in an attenuated inflammasome response that does not result in caspase-1 dependent cell death.

Induced activity in accelerator structures, air and water

CERN Document Server

Stevenson, Graham Roger

2001-01-01

A summary is given of several 'rules of thumb' which can be used to predict the formation and decay of radionuclides in the structure of accelerators together with the dose rates from the induced radioactivity. Models are also given for the activation of gases (air of the accelerator vault) and liquids (in particular cooling water), together with their transport front the activation region to the release point. (18 refs).

Induced activity in accelerator structures, air and water

International Nuclear Information System (INIS)

Stevenson, G.R.

2001-01-01

A summary is given of several 'rules of thumb' which can be used to predict the formation and decay of radionuclides in the structure of accelerators together with the dose rates from the induced radioactivity. Models are also given for the activation of gases (air of the accelerator vault) and liquids (in particular cooling water), together with their transport from the activation region to the release point. (author)

Activation of Protease-Activated Receptor 2 Induces VEGF Independently of HIF-1

DEFF Research Database (Denmark)

Rasmussen, J.G.; Riis, Simone Elkjær; Frøbert, O.

2012-01-01

Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible fact...

Protective Activity of Dendropanax Morbifera Against Cisplatin-Induced Acute Kidney Injury

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Eun-Sun Kim

2015-01-01

Full Text Available Background/Aims: Drug-induced acute kidney injury (AKI has been a severe threat to hospitalized patients, raising the urgent needs to develop strategies to reduce AKI. We investigated the protective activity of Dendropanax morbifera (DP, a medicinal plant which has been widely used to treat infectious and pain diseases, on acute kidney injury (AKI using cisplatin-induced nephropathic models. Methods: Both in vitro renal tubular cells (NRK-52E and in vivo rat models were used to demonstrate the nephroprotective effect of DP. Results: Methanolic extract from DP significantly reduced cisplatin-induced toxicity in renal tubular cells. Through successive liquid extraction, the extract of DP was separated into n-hexane, CHCl3, EtOAc, n-BuOH, and H2O fractions. Among these, the CHCl3 fraction (DPCF was found to be most potent. The protective activity of DPCF was found to be mediated through anti-oxidant, mitochondrial protective, and anti-apoptotic activities. In in vivo rat models of AKI, treatment with DPCF significantly reversed the cisplatin-induced increase in blood urea nitrogen and serum creatinine and histopathologic damage, recovered the level of anti-oxidant enzymes, and inhibited renal apoptosis. Conclusion: We demonstrated that DP extracts decreased cisplatin-induced renal toxicity, indicating its potential to ameliorate drug-associated acute kidney damage.

Code ACTIVE for calculation of the transmutation, induced activity and decay heat in neutron irradiation

International Nuclear Information System (INIS)

Ioki, Kimihiro; Harada, Yuhei; Asami, Naoto.

1976-03-01

The computer code ACTIVE has been prepared for calculation of the transmutation rate, induced activity and decay heat. Calculations are carried out with activation chain and spatial distribution of neutron energy spectrum. The spatial distribution of secondary gamma-ray source due to the unstable nuclides is also obtainable. Special attension is paid to the short life decays. (auth.)

Estradiol-induced antinociceptive responses on formalin-induced nociception are independent of COX and HPA activation.

Science.gov (United States)

Hunter, Deirtra A; Barr, Gordon A; Amador, Nicole; Shivers, Kai-Yvonne; Kemen, Lynne; Kreiter, Christopher M; Jenab, Shirzad; Inturrisi, Charles E; Quinones-Jenab, Vanya

2011-07-01

Estrogen modulates pain perception but how it does so is not fully understood. The aim of this study was to determine if estradiol reduces nociceptive responses in part via hypothalamic-pituitary-adrenal (HPA) axis regulation of cyclooxygenase (COX)-1/COX-2 activity. The first study examined the effects of estradiol (20%) or vehicle with concurrent injection nonsteroidal antiinflammatory drugs (NSAIDs) on formalin-induced nociceptive responding (flinching) in ovariectomized (OVX) rats. The drugs were ibuprofen (COX-1 and COX-2 inhibitor), SC560 (COX-1 inhibitor), or NS398 (COX-2 inhibitor). In a second study, estradiol's effects on formalin-induced nociception were tested in adrenalectomized (ADX), OVX, and ADX+OVX rats. Serum levels of prostaglandins (PG) PGE(2) and corticosterone were measured. Estradiol significantly decreased nociceptive responses in OVX rats with effects during both the first and the second phase of the formalin test. The nonsteroidal antiinflammatory drugs (NSAIDs) did not alter nociception at the doses used here. Adrenalectomy neither altered flinching responses in female rats nor reversed estradiol-induced antinociceptive responses. Estradiol alone had no effect on corticosterone (CORT) or prostaglandin levels after the formalin test, dissociating the effects of estradiol on behavior and these serum markers. Ibuprofen and NS398 significantly reduced PGE2 levels. CORT was not decreased by OVX surgery or by estradiol below that of ADX. Only IBU significantly increased corticosterone levels. Taken together, our results suggest that estradiol-induced antinociception in female rats is independent of COX activity and HPA axis activation. Copyright © 2010 Wiley-Liss, Inc.

Hepatitis B virus e antigen induces activation of rat hepatic stellate cells

International Nuclear Information System (INIS)

Zan, Yanlu; Zhang, Yuxia; Tien, Po

2013-01-01

Highlights: •HBeAg expression in HSCs induced production of ECM protein and liver fibrotic markers. •The activation and proliferation of HSCs were mediated by TGF-β. •HBeAg protein purified from cell medium directly activated HSCs. -- Abstract: Chronic hepatitis B virus infection is a major cause of hepatic fibrosis, leading to liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus e antigen (HBeAg) is an accessory protein of HBV, not required for viral replication but important for natural infection in vivo. Hepatic stellate cells (HSCs) are the major producers of excessive extracellular matrix during liver fibrogenesis. Therefore, we examined the influence of HBeAg on HSCs. The rat HSC line HSC-T6 was transfected with HBeAg plasmids, and expression of α-smooth muscle actin, collagen I, transforming growth factor-β1 (TGF-β), and tissue inhibitors of metalloproteinase 1 (TIMP-1) was investigated by quantitative real-time PCR. The proliferation of HSCs was determined by MTS analysis. HBeAg transduction induced up-regulation of these fibrogenic genes and proliferation of HSCs. We found that HBeAg induced TGF-β secretion in HSCs, and the activation of HSCs was prevented by a neutralizing anti-TGF-β antibody. Depletion and addition of HBeAg protein in conditioned medium from HSC-T6 cells transduced with HBeAg indicated that HBeAg directly induced the activation and proliferation of rat primary HSCs. Taken together, HBeAg induces the activation and proliferation of HSCs, mainly mediated by TGF-β, and HBeAg protein purified from cell medium can directly activate HSCs

Hepatitis B virus e antigen induces activation of rat hepatic stellate cells

Energy Technology Data Exchange (ETDEWEB)

Zan, Yanlu [Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Zhang, Yuxia, E-mail: yzhang@wehi.edu.au [Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China); Tien, Po, E-mail: tienpo@sun.im.ac.cn [Center for Molecular Virology, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101 (China)

2013-06-07

Highlights: •HBeAg expression in HSCs induced production of ECM protein and liver fibrotic markers. •The activation and proliferation of HSCs were mediated by TGF-β. •HBeAg protein purified from cell medium directly activated HSCs. -- Abstract: Chronic hepatitis B virus infection is a major cause of hepatic fibrosis, leading to liver cirrhosis and hepatocellular carcinoma. Hepatitis B virus e antigen (HBeAg) is an accessory protein of HBV, not required for viral replication but important for natural infection in vivo. Hepatic stellate cells (HSCs) are the major producers of excessive extracellular matrix during liver fibrogenesis. Therefore, we examined the influence of HBeAg on HSCs. The rat HSC line HSC-T6 was transfected with HBeAg plasmids, and expression of α-smooth muscle actin, collagen I, transforming growth factor-β1 (TGF-β), and tissue inhibitors of metalloproteinase 1 (TIMP-1) was investigated by quantitative real-time PCR. The proliferation of HSCs was determined by MTS analysis. HBeAg transduction induced up-regulation of these fibrogenic genes and proliferation of HSCs. We found that HBeAg induced TGF-β secretion in HSCs, and the activation of HSCs was prevented by a neutralizing anti-TGF-β antibody. Depletion and addition of HBeAg protein in conditioned medium from HSC-T6 cells transduced with HBeAg indicated that HBeAg directly induced the activation and proliferation of rat primary HSCs. Taken together, HBeAg induces the activation and proliferation of HSCs, mainly mediated by TGF-β, and HBeAg protein purified from cell medium can directly activate HSCs.

Targeting cytokine signaling checkpoint CIS activates NK cells to protect from tumor initiation and metastasis

Science.gov (United States)

Putz, Eva M.; Guillerey, Camille; Kos, Kevin; Stannard, Kimberley; Miles, Kim; Delconte, Rebecca B.; Nicholson, Sandra E.; Huntington, Nicholas D.; Smyth, Mark J.

2017-01-01

ABSTRACT The cytokine-induced SH2-containing protein CIS belongs to the suppressor of cytokine signaling (SOCS) protein family. Here, we show the critical role of CIS in suppressing natural killer (NK) cell control of tumor initiation and metastasis. Cish-deficient mice were highly resistant to methylcholanthrene-induced sarcoma formation and protected from lung metastasis of B16F10 melanoma and RM-1 prostate carcinoma cells. In contrast, the growth of primary subcutaneous tumors, including those expressing the foreign antigen OVA, was unchanged in Cish-deficient mice. The combination of Cish deficiency and relevant targeted and immuno-therapies such as combined BRAF and MEK inhibitors, immune checkpoint blockade antibodies, IL-2 and type I interferon revealed further improved control of metastasis. The data clearly indicate that targeting CIS promotes NK cell antitumor functions and CIS holds great promise as a novel target in NK cell immunotherapy. PMID:28344878

Ouabain exacerbates activation-induced cell death in human peripheral blood lymphocytes

OpenAIRE

Esteves Mabel B.; Marques-Santos Luis F.; Affonso-Mitidieri Ottília R.; Rumjanek Vivian M.

2005-01-01

Lymphocytes activated by mitogenic lectins display changes in transmembrane potential, an elevation in the cytoplasmic Ca2+ concentrations, proliferation and/or activation induced cell death. Low concentrations of ouabain (an inhibitor of Na+,K+-ATPase) suppress mitogen-induced proliferation and increases cell death. To understand the mechanisms involved, a number of parameters were analyzed using fluorescent probes and flow cytometry. The addition of 100nM ouabain to cultures of peripheral b...

Brucella abortus-activated microglia induce neuronal death through primary phagocytosis.

Science.gov (United States)

Rodríguez, Ana M; Delpino, M Victoria; Miraglia, M Cruz; Costa Franco, Miriam M; Barrionuevo, Paula; Dennis, Vida A; Oliveira, Sergio C; Giambartolomei, Guillermo H

2017-07-01

Inflammation has long been implicated as a contributor to pathogenesis in neurobrucellosis. Many of the associated neurocognitive symptoms of neurobrucellosis may be the result of neuronal dysfunction resulting from the inflammatory response induced by Brucella abortus infection in the central nervous system. In this manuscript, we describe an immune mechanism for inflammatory activation of microglia that leads to neuronal death upon B. abortus infection. B. abortus was unable to infect or harm primary cultures of mouse neurons. However, when neurons were co-cultured with microglia and infected with B. abortus significant neuronal loss occurred. This phenomenon was dependent on TLR2 activation by Brucella lipoproteins. Neuronal death was not due to apoptosis, but it was dependent on the microglial release of nitric oxide (NO). B. abortus infection stimulated microglial proliferation, phagocytic activity and engulfment of neurons. NO secreted by B. abortus-activated microglia induced neuronal exposure of the "eat-me" signal phosphatidylserine (PS). Blocking of PS-binding to protein milk fat globule epidermal growth factor-8 (MFG-E8) or microglial vitronectin receptor-MFG-E8 interaction was sufficient to prevent neuronal loss by inhibiting microglial phagocytosis without affecting their activation. Taken together, our results indicate that B. abortus is not directly toxic to neurons; rather, these cells become distressed and are killed by phagocytosis in the inflammatory surroundings generated by infected microglia. Neuronal loss induced by B. abortus-activated microglia may explain, in part, the neurological deficits observed during neurobrucellosis. © 2017 Wiley Periodicals, Inc.

Sequential activation of proteases in radiation induced apoptosis

International Nuclear Information System (INIS)

Watters, D.; Waterhouse, N.

1997-01-01

Full text: Significant advances have been made in recent years in unraveling the molecular mechanisms of apoptosis particularly in relation to Fas- and TNF-mediated cell death, however there are considerable gaps in our knowledge of the processes involved in apoptosis induced by ionizing radiation. We have used the degradation of specific proteolytic targets in a pair of isogenic Burkitt's Iymphoma cells lines (BL30A, sensitive and BL30K resistant) to study the sequence of events in the execution of radiation-induced apoptosis. Fodrin can be cleaved to fragments of 150 kDa and 120 kDa. In the case of Fas-mediated apoptosis both cleavages are inhibited by the caspase inhibitor zVAD-fmk at 10 μM, a concentration which inhibits all the hallmarks of apoptosis. However in radiation-induced apoptosis, inhibition of the clevage of fodrin to the 150 kDa fragment requires 100 μM zVAD-fink while apoptosis itself is inhibited at 10 μM. This suggests that different enzymes are responsible for the generation of the 150 kDa fragment in the two models of apoptosis. Fodrin has been reported to be cleaved by μ-calpain to a 150 kDa fragment however, the involvement of μ-calpain in apoptosis has not yet been established. In murine fodrin there is a caspase cleavage site within 1 kDa of the calpain cleavage site. In vitro studies using purified enzymes showed that only caspase-3 and μ-calpain could cleave fodrin in untreated cell extracts to the same sized fragments as seen during apoptosis in vivo. We provide evidence for the early activation of μ-calpain after ionizing radiation in the sensitive BL30A cell line, and show that the time course of μ-calpain activation parallels that of the appearance of the 150 kDa fragment. Caspase-3 is activated much later and is likely to be responsible for the generation of the 120 kDa fragment. μ-Calpain was not activated in the resistant cell line. Based on these results we propose a model for the proteolytic cascade in radiation-induced

Roles of acid sphingomyelinase activation in neuronal cells apoptosis induced by microwave irradiation

International Nuclear Information System (INIS)

Zhang Lei; Xu Shangcheng; Zhang Guangbin; Yu Zhengping

2009-01-01

The present study is to examine the effect of microwave on acid sphingomyelinase (ASM) activity and expression, and to explore the role of ASM activation in neuronal cells apoptosis induced by microwave irradiation. Primary cultured hippocampal neurons were irradiated by 30 W/cm 2 microwave for 10 min, and ASM activity assay was used to investigate ASM activity alteration. RT-PCR and western blot were used to detect ASM mRNA and protein expression respectively. Apoptosis was observed by Hoechst 33342 fluorescence staining. ASM specific inhibitor imipramine was applied to inhibit ASM activation. It has been found that apoptosis rate of primary cultured hippocampal neurons increased significantly after microwave irradiation. ASM was activated while ASM mRNA and protein expression were upregulated in neurons after microwave irradiation. Pretreatment with imipramine could reverse neuronal apoptosis induced by microwave irradiation. Results show that microwave irradiation causes increment of ASM activation and expression and ASM activation is involved in microwave induced neuronal apoptosis. (authors)

PPARβ/δ regulates glucocorticoid- and sepsis-induced FOXO1 activation and muscle wasting.

Directory of Open Access Journals (Sweden)

Estibaliz Castillero

Full Text Available FOXO1 is involved in glucocorticoid- and sepsis-induced muscle wasting, in part reflecting regulation of atrogin-1 and MuRF1. Mechanisms influencing FOXO1 expression in muscle wasting are poorly understood. We hypothesized that the transcription factor peroxisome proliferator-activated receptor β/δ (PPARβ/δ upregulates muscle FOXO1 expression and activity with a downstream upregulation of atrogin-1 and MuRF1 expression during sepsis and glucocorticoid treatment and that inhibition of PPARβ/δ activity can prevent muscle wasting. We found that activation of PPARβ/δ in cultured myotubes increased FOXO1 activity, atrogin-1 and MuRF1 expression, protein degradation and myotube atrophy. Treatment of myotubes with dexamethasone increased PPARβ/δ expression and activity. Dexamethasone-induced FOXO1 activation and atrogin-1 and MuRF1 expression, protein degradation, and myotube atrophy were inhibited by PPARβ/δ blocker or siRNA. Importantly, muscle wasting induced in rats by dexamethasone or sepsis was prevented by treatment with a PPARβ/δ inhibitor. The present results suggest that PPARβ/δ regulates FOXO1 activation in glucocorticoid- and sepsis-induced muscle wasting and that treatment with a PPARβ/δ inhibitor may ameliorate loss of muscle mass in these conditions.

Growth hormone-induced insulin resistance in human subjects involves reduced pyruvate dehydrogenase activity

DEFF Research Database (Denmark)

Nellemann, B.; Vendelbo, M.H.; Nielsen, Thomas Svava

2014-01-01

Insulin resistance induced by growth hormone (GH) is linked to promotion of lipolysis by unknown mechanisms. We hypothesized that suppression of the activity of pyruvate dehydrogenase in the active form (PDHa) underlies GH-induced insulin resistance similar to what is observed during fasting....

Substrate Stiffness Influences Doxorubicin-Induced p53 Activation via ROCK2 Expression

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Takahiro Ebata

2017-01-01

Full Text Available The physical properties of the extracellular matrix (ECM, such as stiffness, are involved in the determination of the characteristics of cancer cells, including chemotherapy sensitivity. Resistance to chemotherapy is often linked to dysfunction of tumor suppressor p53; however, it remains elusive whether the ECM microenvironment interferes with p53 activation in cancer cells. Here, we show that, in MCF-7 breast cancer cells, extracellular stiffness influences p53 activation induced by the antitumor drug doxorubicin. Cell growth inhibition by doxorubicin was increased in response to ECM rigidity in a p53-dependent manner. The expression of Rho-associated coiled coil-containing protein kinase (ROCK 2, which induces the activation of myosin II, was significantly higher when cells were cultured on stiffer ECM substrates. Knockdown of ROCK2 expression or pharmacological inhibition of ROCK decreased doxorubicin-induced p53 activation. Our results suggest that a soft ECM causes downregulation of ROCK2 expression, which drives resistance to chemotherapy by repressing p53 activation.

Kainate-induced network activity in the anterior cingulate cortex.

Science.gov (United States)

Shinozaki, R; Hojo, Y; Mukai, H; Hashizume, M; Murakoshi, T

2016-06-14

Anterior cingulate cortex (ACC) plays a pivotal role in higher order processing of cognition, attention and emotion. The network oscillation is considered an essential means for integration of these CNS functions. The oscillation power and coherence among related areas are often dis-regulated in several psychiatric and pathological conditions with a hemispheric asymmetric manner. Here we describe the network-based activity of field potentials recorded from the superficial layer of the mouse ACC in vitro using submerged type recordings. A short activation by kainic acid administration to the preparation induced populational activities ranging over several frequency bands including theta (3-8Hz), alpha (8-12Hz), beta (13-30Hz), low gamma (30-50Hz) and high gamma (50-80Hz). These responses were repeatable and totally abolished by tetrodotoxin, and greatly diminished by inhibitors of ionotropic and metabotropic glutamate receptors, GABAA receptor or gap-junctions. These observations suggest that the kainate-induced network activity can be a useful model of the network oscillation in the ACC circuit. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Fisetin, a dietary flavonoid, augments the anti-invasive and anti-metastatic potential of sorafenib in melanoma.

Science.gov (United States)

Pal, Harish C; Diamond, Ariana C; Strickland, Leah R; Kappes, John C; Katiyar, Santosh K; Elmets, Craig A; Athar, Mohammad; Afaq, Farrukh

2016-01-12

Melanoma is the most aggressive and deadly form of cutaneous neoplasm due to its propensity to metastasize. Oncogenic BRAF drives sustained activation of the BRAF/MEK/ERK (MAPK) pathway and cooperates with PI3K/AKT/mTOR (PI3K) signaling to induce epithelial to mesenchymal transition (EMT), leading to cell invasion and metastasis. Therefore, targeting these pathways is a promising preventive/therapeutic strategy. We have shown that fisetin, a flavonoid, reduces human melanoma cell invasion by inhibiting EMT. In addition, fisetin inhibited melanoma cell proliferation and tumor growth by downregulating the PI3K pathway. In this investigation, we aimed to determine whether fisetin can potentiate the anti-invasive and anti-metastatic effects of sorafenib in BRAF-mutated melanoma. We found that combination treatment (fisetin + sorafenib) more effectively reduced the migration and invasion of BRAF-mutated melanoma cells both in vitro and in raft cultures compared to individual agents. Combination treatment also effectively inhibited EMT as observed by a decrease in N-cadherin, vimentin and fibronectin and an increase in E-cadherin both in vitro and in xenograft tumors. Furthermore, combination therapy effectively inhibited Snail1, Twist1, Slug and ZEB1 protein expression compared to monotherapy. The expression of MMP-2 and MMP-9 in xenograft tumors was further reduced in combination treatment compared to individual agents. Bioluminescent imaging of athymic mice, intravenously injected with stably transfected CMV-luciferase-ires-puromycin.T2A.EGFP-tagged A375 melanoma cells, demonstrated fewer lung metastases following combination treatment versus monotherapy. Our findings demonstrate that fisetin potentiates the anti-invasive and anti-metastatic effects of sorafenib. Our data suggest that fisetin may be a worthy adjuvant chemotherapy for the management of melanoma.

Artemisinin induces ROS-mediated caspase3 activation in ASTC-a-1 cells

Science.gov (United States)

Xiao, Feng-Lian; Chen, Tong-Sheng; Qu, Jun-Le; Liu, Cheng-Yi

2010-02-01

Artemisinin (ART), an antimalarial phytochemical from the sweet wormwood plant or a naturally occurring component of Artemisia annua, has been shown a potential anticancer activity by apoptotic pathways. In our report, cell counting kit (CCK-8) assay showed that treatment of human lung adenocarcinoma (ASTC-a-1) cells with ART effectively increase cell death by inducing apoptosis in a time- and dose-dependent fashion. Hoechst 33258 staining was used to detect apoptosis as well. Reactive oxygen species (ROS) generation was observed in cells exposed to ART at concentrations of 400 μM for 48 h. N-acetyl-L-cysteine (NAC), an oxygen radical scavenger, suppressed the rate of ROS generation and inhibited the ART-induced apoptosis. Moreover, AFC assay (Fluorometric assay for Caspase3 activity) showed that ROS was involved in ART-induced caspase3 acitvation. Taken together, our data indicate that ART induces ROS-mediated caspase3 activation in a time-and dose-dependent way in ASCT-a-1 cells.

Involvement of microglia activation in the lead induced long-term potentiation impairment.

Directory of Open Access Journals (Sweden)

Ming-Chao Liu

Full Text Available Exposure of Lead (Pb, a known neurotoxicant, can impair spatial learning and memory probably via impairing the hippocampal long-term potentiation (LTP as well as hippocampal neuronal injury. Activation of hippocampal microglia also impairs spatial learning and memory. Thus, we raised the hypothesis that activation of microglia is involved in the Pb exposure induced hippocampal LTP impairment and neuronal injury. To test this hypothesis and clarify its underlying mechanisms, we investigated the Pb-exposure on the microglia activation, cytokine release, hippocampal LTP level as well as neuronal injury in in vivo or in vitro model. The changes of these parameters were also observed after pretreatment with minocycline, a microglia activation inhibitor. Long-term low dose Pb exposure (100 ppm for 8 weeks caused significant reduction of LTP in acute slice preparations, meanwhile, such treatment also significantly increased hippocampal microglia activation as well as neuronal injury. In vitro Pb-exposure also induced significantly increase of microglia activation, up-regulate the release of cytokines including tumor necrosis factor-alpha (TNF-α, interleukin-1β (IL-1β and inducible nitric oxide synthase (iNOS in microglia culture alone as well as neuronal injury in the co-culture with hippocampal neurons. Inhibiting the microglia activation with minocycline significantly reversed the above-mentioned Pb-exposure induced changes. Our results showed that Pb can cause microglia activation, which can up-regulate the level of IL-1β, TNF-α and iNOS, these proinflammatory factors may cause hippocampal neuronal injury as well as LTP deficits.

Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: Molecular mechanisms and barrier-protective role.

Science.gov (United States)

Kovacs-Kasa, Anita; Kim, Kyung Mi; Cherian-Shaw, Mary; Black, Stephen M; Fulton, David J; Verin, Alexander D

2018-08-01

We have previously shown that Gs-coupled adenosine receptors (A2a) are primarily involved in adenosine-induced human pulmonary artery endothelial cell (HPAEC) barrier enhancement. However, the downstream events that mediate the strengthening of the endothelial cell (EC) barrier via adenosine signaling are largely unknown. In the current study, we tested the overall hypothesis that adenosine-induced Rac1 activation and EC barrier enhancement is mediated by Gs-dependent stimulation of cAMP-dependent Epac1-mediated signaling cascades. Adenoviral transduction of HPAEC with constitutively-active (C/A) Rac1 (V12Rac1) significantly increases transendothelial electrical resistance (TER) reflecting an enhancement of the EC barrier. Conversely, expression of an inactive Rac1 mutant (N17Rac1) decreases TER reflecting a compromised EC barrier. The adenosine-induced increase in TER was accompanied by activation of Rac1, decrease in contractility (MLC dephosphorylation), but not Rho inhibition. Conversely, inhibition of Rac1 activity attenuates adenosine-induced increase in TER. We next examined the role of cAMP-activated Epac1 and its putative downstream targets Rac1, Vav2, Rap1, and Tiam1. Depletion of Epac1 attenuated the adenosine-induced Rac1 activation and the increase in TER. Furthermore, silencing of Rac1 specific guanine nucleotide exchange factors (GEFs), Vav2 and Rap1a expression significantly attenuated adenosine-induced increases in TER and activation of Rac1. Depletion of Rap1b only modestly impacted adenosine-induced increases in TER and Tiam1 depletion had no effect on adenosine-induced Rac1 activation and TER. Together these data strongly suggest that Rac1 activity is required for adenosine-induced EC barrier enhancement and that the activation of Rac1 and ability to strengthen the EC barrier depends, at least in part, on cAMP-dependent Epac1/Vav2/Rap1-mediated signaling. © 2017 Wiley Periodicals, Inc.

Minocycline attenuates sevoflurane-induced cell injury via activation of Nrf2.

Science.gov (United States)

Tian, Yue; Wu, Xiuying; Guo, Shanbin; Ma, Ling; Huang, Wei; Zhao, Xiaochun

2017-04-01

Minocycline has been demonstrated to exert neuroprotective effects in various experimental models. In the present study, we investigated the mechanisms underlying the protective effects of minocycline on cell injury induced by the inhalation of the anesthetic, sevoflurane. In our in vivo experiments using rats, minocycline attenuated sevoflurane-induced neuronal degeneration and apoptosis in the rat hippocampus, and this effect was associated with the minocycline-mediated suppression of oxidative stress in the hippocampus. In in vitro experiments, minocycline inhibited sevoflurane-induced apoptosis and the production of reactive oxygen species (ROS) in H4 human neuroglioma cells. In addition, minocycline suppressed the sevoflurane-induced upregulation of interleukin (IL)-6 and the activation of the nuclear factor-κB (NF-κB) signaling pathway in H4 cells. Furthermore, we found that nuclear factor E2-related factor 2 (Nrf2), an activator of the stress response, was upregulated and activated upon sevoflurane treatment both in the rat hippocampus and in H4 cells. In addition, minocycline further augmented the upregulation and activation of Nrf2 when used in conjunction with sevoflurane. Moreover, the knockdown of Nrf2 in H4 cells by small interfering RNA (siRNA) diminished the cytoprotective effect of minocycline, and attenuated the inhibitory effect of minocycline on ROS production, IL-6 upregulation and the activation of the NF-κB signaling pathway. On the whole, our findings indicate that minocycline may exert protective effects against sevoflurane-induced cell injury via the Nrf2-modulated antioxidant response and the inhibition of the activation of the NF-κB signaling pathway.

Active-interrogation measurements of fast neutrons from induced fission in low-enriched uranium

International Nuclear Information System (INIS)

Dolan, J.L.; Marcath, M.J.; Flaska, M.; Pozzi, S.A.; Chichester, D.L.; Tomanin, A.; Peerani, P.

2014-01-01

A detection system was designed with MCNPX-PoliMi to measure induced-fission neutrons from U-235 and U-238 using active interrogation. Measurements were then performed with this system at the Joint Research Centre in Ispra, Italy on low-enriched uranium samples. Liquid scintillators measured induced fission neutrons to characterize the samples in terms of their uranium mass and enrichment. Results are presented to investigate and support the use of organic liquid scintillators with active interrogation techniques to characterize uranium containing materials. -- Highlights: • We studied low-enriched uranium using active-interrogation experiments including a deuterium–tritium neutron generator and an americium–lithium isotopic neutron source. • Liquid scintillators measured induced-fission neutrons from the active-interrogation methods. • Fast-neutron (DT) and thermal-neutron (Am–Li) interrogation resulted in the measurement of trends in uranium mass and 235 U enrichment respectively. • MCNPX-PoliMi, the Monte Carlo transport code, simulated the measured induced-fission neutron trends in the liquid scintillators

Active-interrogation measurements of fast neutrons from induced fission in low-enriched uranium

Energy Technology Data Exchange (ETDEWEB)

Dolan, J.L., E-mail: jldolan@umich.edu [Department of Nuclear Engineering and Radiological Sciences, University of Michigan, Ann Arbor, MI 48109 (United States); Marcath, M.J.; Flaska, M.; Pozzi, S.A. [Department of Nuclear Engineering and Radiological Sciences, University of Michigan, Ann Arbor, MI 48109 (United States); Chichester, D.L. [Idaho National Laboratory, Idaho Falls, ID 83415 (United States); Tomanin, A.; Peerani, P. [European Commission, Joint Research Centre, Institute for Transuranium Elements, Ispra (Italy)

2014-02-21

A detection system was designed with MCNPX-PoliMi to measure induced-fission neutrons from U-235 and U-238 using active interrogation. Measurements were then performed with this system at the Joint Research Centre in Ispra, Italy on low-enriched uranium samples. Liquid scintillators measured induced fission neutrons to characterize the samples in terms of their uranium mass and enrichment. Results are presented to investigate and support the use of organic liquid scintillators with active interrogation techniques to characterize uranium containing materials. -- Highlights: • We studied low-enriched uranium using active-interrogation experiments including a deuterium–tritium neutron generator and an americium–lithium isotopic neutron source. • Liquid scintillators measured induced-fission neutrons from the active-interrogation methods. • Fast-neutron (DT) and thermal-neutron (Am–Li) interrogation resulted in the measurement of trends in uranium mass and {sup 235}U enrichment respectively. • MCNPX-PoliMi, the Monte Carlo transport code, simulated the measured induced-fission neutron trends in the liquid scintillators.

Troglitazone induced apoptosis via PPARγ activated POX-induced ROS formation in HT29 cells.

Science.gov (United States)

Wang, Jing; Lv, XiaoWen; Shi, JiePing; Hu, XiaoSong; DU, YuGuo

2011-08-01

In order to investigate the potential mechanisms in troglitazone-induced apoptosis in HT29 cells, the effects of PPARγ and POX-induced ROS were explored. [3- (4, 5)-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, Annexin V and PI staining using FACS, plasmid transfection, ROS formation detected by DCFH staining, RNA interference, RT-PCR & RT-QPCR, and Western blotting analyses were employed to investigate the apoptotic effect of troglitazone and the potential role of PPARγ pathway and POX-induced ROS formation in HT29 cells. Troglitazone was found to inhibit the growth of HT29 cells by induction of apoptosis. During this process, mitochondria related pathways including ROS formation, POX expression and cytochrome c release increased, which were inhibited by pretreatment with GW9662, a specific antagonist of PPARγ. These results illustrated that POX upregulation and ROS formation in apoptosis induced by troglitazone was modulated in PPARγ-dependent pattern. Furthermore, the inhibition of ROS and apoptosis after POX siRNA used in troglitazone-treated HT29 cells indicated that POX be essential in the ROS formation and PPARγ-dependent apoptosis induced by troglitazone. The findings from this study showed that troglitazone-induced apoptosis was mediated by POX-induced ROS formation, at least partly, via PPARγ activation. Copyright © 2011 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.

Role of Bioavailable Iron in Coal Dust-Induced Activation of Activator Protein-1 and Nuclear Factor of Activated T Cells

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Huang, Chuanshu; Li, Jingxia; Zhang, Qi; Huang, Xi

2010-01-01

Activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) are two important transcription factors responsible for the regulation of cytokines, which are involved in cell proliferation and inflammation. Coal workers’ pneumoconiosis (CWP) is an occupational lung disease that may be related to chronic inflammation caused by coal dust exposure. In the present study, we demonstrate that coal from the Pennsylvania (PA) coalmine region, which has a high prevalence of CWP, can activate both AP-1 and NFAT in JB6 mouse epidermal cells. In contrast, coal from the Utah (UT) coalmine region, which has a low prevalence of CWP, has no such effects. The PA coal stimulates mitogen-activated protein kinase (MAPK) family members of extracellular signal-regulated kinases (ERKs) and p38 MAPK but not c-Jun-NH2-terminal kinases, as determined by the phosphorylation assay. The increase in AP-1 by the PA coal was completely eliminated by the pretreatment of cells with PD98059, a specific MAPK kinase inhibitor, and SB202190, a p38 kinase inhibitor, further confirming that the PA coal-induced AP-1 activation is mediated through ERKs and p38 MAPK pathways. Deferoxamine (DFO), an iron chelator, synergistically enhanced the PA coal-induced AP-1 activity, but inhibited NFAT activity. For comparison, cells were treated with ferrous sulfate and/or DFO. We have found that iron transactivated both AP-1 and NFAT, and DFO further enhanced iron-induced AP-1 activation but inhibited NFAT. These results indicate that activation of AP-1 and NFAT by the PA coal is through bioavailable iron present in the coal. These data are in agreement with our previous findings that the prevalence of CWP correlates well with levels of bioavailable iron in coals from various mining regions. PMID:12397016

Natural Product Vibsanin A Induces Differentiation of Myeloid Leukemia Cells through PKC Activation.

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Yu, Zu-Yin; Xiao, He; Wang, Li-Mei; Shen, Xing; Jing, Yu; Wang, Lin; Sun, Wen-Feng; Zhang, Yan-Feng; Cui, Yu; Shan, Ya-Jun; Zhou, Wen-Bing; Xing, Shuang; Xiong, Guo-Lin; Liu, Xiao-Lan; Dong, Bo; Feng, Jian-Nan; Wang, Li-Sheng; Luo, Qing-Liang; Zhao, Qin-Shi; Cong, Yu-Wen

2016-05-01

All-trans retinoic acid (ATRA)-based cell differentiation therapy has been successful in treating acute promyelocytic leukemia, a unique subtype of acute myeloid leukemia (AML). However, other subtypes of AML display resistance to ATRA-based treatment. In this study, we screened natural, plant-derived vibsane-type diterpenoids for their ability to induce differentiation of myeloid leukemia cells, discovering that vibsanin A potently induced differentiation of AML cell lines and primary blasts. The differentiation-inducing activity of vibsanin A was mediated through direct interaction with and activation of protein kinase C (PKC). Consistent with these findings, pharmacological blockade of PKC activity suppressed vibsanin A-induced differentiation. Mechanistically, vibsanin A-mediated activation of PKC led to induction of the ERK pathway and decreased c-Myc expression. In mouse xenograft models of AML, vibsanin A administration prolonged host survival and inhibited PKC-mediated inflammatory responses correlated with promotion of skin tumors in mice. Collectively, our results offer a preclinical proof of concept for vibsanin A as a myeloid differentiation-inducing compound, with potential application as an antileukemic agent. Cancer Res; 76(9); 2698-709. ©2016 AACR. ©2016 American Association for Cancer Research.

Creatine kinase activity in dogs with experimentally induced acute inflammation

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Dimitrinka Zapryanova

2013-01-01

Full Text Available The main purpose of this study was to investigate the effect of acute inflammation on total creatine kinase (CK activity in dogs. In these animals, CK is an enzyme found predominantly in skeletal muscle and significantly elevated serum activity is largely associated with muscle damage. Plasma increases in dogs are associated with cell membrane leakage and will therefore be seen in any condition associated with muscular inflammation. The study was induced in 15 mongrel male dogs (n=9 in experimental group and n=6 in control group at the age of two years and body weight 12-15 kg. The inflammation was reproduced by inoculation of 2 ml turpentine oil subcutaneously in lumbar region. The plasma activity of creatine kinase was evaluated at 0, 6, 24, 48, 72 hours after inoculation and on days 7, 14 and 21 by a kit from Hospitex Diagnostics. In the experimental group, the plasma concentrations of the CK-activity were increased at the 48th hour (97.48±6.92 U/L and remained significantly higher (p<0.05 at the 72 hour (97.43±2.93 U/L compared to the control group (77.08±5.27 U/L. The results of this study suggest that the evaluation of creatine kinase in dogs with experimentally induced acute inflammation has a limited diagnostic value. It was observed that the creatine kinase activity is slightly affected by the experimentally induced acute inflammation in dogs.

Modern g,d,p,n-induced activation-transmutation systems

International Nuclear Information System (INIS)

Sublet, J.Ch.

2010-01-01

Document available in abstract form only. Full text of publication follows: The European Activation System (EASY) includes as the source of nuclear data the European Activation File (EAF) and as its engine the FISPACT activation-transmutation code. The latest version of the EAF, EAF-2010, contains cross-section data for gamma-, deuteron- and proton-induced reactions in addition to the traditional neutron-induced data. The main reason for the addition of these data to EAF is to enable activation-transmutation calculations to be performed for even more nuclear facilities, including 'accelerator'-driven devices with incident upper energy limit of 60 or 200 MeV. EAF-2010 has benefited from the generation and maintenance of comprehensive activation files in the past and the development of the processing code SAFEPAQ-II and model code TALYS. TALYS is the source for all gamma-, proton- and deuteron-induced data and a fair share of the neutron-induced data. Cross-section validation exercises against both experimental data and systematic, which were started in 1995, enable a comprehensive assessment of the data. Although EAF-2010 is certainly the most-validated activation neutron cross-section library in the world, currently less than 3% of all the reactions can be compared with experimental information, and even then only for a very limited, and not always application-relevant, energy range. As with EAF-2010, -2003, -2005 and -2007 results of integral experiments have been used to correct, adjust and validate data. This can be done using SAFEPAQ II by inputting the measured effective cross-sections. Validation using integral data has been performed by means of direct comparison with measurements of various materials under relevant particle spectra. A tool has recently been developed which is important now that the libraries contain so much TALYS-calculated data. Statistical analysis of cross-sections (SACS) is used to look for trends in the library data for a particular

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

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Fang Rao

Full Text Available Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ. We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg or increased (120, 180, 240 mmHg hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg. The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs. These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

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Rao, Fang; Yang, Ren-Qiang; Chen, Xiao-Shu; Xu, Jin-Song; Fu, Hui-Min; Su, Hai; Wang, Ling

2014-01-01

Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ). We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg) or increased (120, 180, 240 mmHg) hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa) binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L) was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg). The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs). These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

Anti-apoptotic BFL-1 is the major effector in activation-induced human mast cell survival.

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Maria Ekoff

Full Text Available Mast cells are best known for their role in allergic reactions, where aggregation of FcεRI leads to the release of mast cell mediators causing allergic symptoms. The activation also induces a survival program in the cells, i.e., activation-induced mast cell survival. The aim of the present study was to investigate how the activation-induced survival is mediated. Cord blood-derived mast cells and the mast cell line LAD-2 were activated through FcεRI crosslinking, with or without addition of chemicals that inhibit the activity or expression of selected Bcl-2 family members (ABT-737; roscovitine. Cell viability was assessed using staining and flow cytometry. The expression and function of Bcl-2 family members BFL-1 and MCL-1 were investigated using real-time quantitative PCR and siRNA treatment. The mast cell expression of Bfl-1 was investigated in skin biopsies. FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1. ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L, Bcl-2, Bcl-w and Mcl-1. Reducing BFL-1 but not MCL-1 levels by siRNA inhibited activation-induced mast cell survival. We also demonstrate that mast cell expression of Bfl-1 is elevated in birch-pollen-provocated skin and in lesions of atopic dermatitis and psoriasis patients. Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival.

CD36 participates in PrP(106-126-induced activation of microglia.

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Mohammed Kouadir

Full Text Available Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The molecular mechanisms that underlie prion-induced microglial activation are not very well understood. In the present study, we investigated the role of the class B scavenger receptor CD36 in microglial activation induced by neurotoxic prion protein (PrP fragment 106-126 (PrP(106-126. We first examined the time course of CD36 mRNA expression upon exposure to PrP(106-126 in BV2 microglia. We then analyzed different parameters of microglial activation in PrP(106-126-treated cells in the presence or not of anti-CD36 monoclonal antibody (mAb. The cells were first incubated for 1 h with CD36 monoclonal antibody to block the CD36 receptor, and were then treated with neurotoxic prion peptides PrP(106-126. The results showed that PrP(106-126 treatment led to a rapid yet transitory increase in the mRNA expression of CD36, upregulated mRNA and protein levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α, increased iNOS expression and nitric oxide (NO production, stimulated the activation of NF-κB and caspase-1, and elevated Fyn activity. The blockade of CD36 had no effect on PrP(106-126-stimulated NF-κB activation and TNF-α protein release, abrogated the PrP(106-126-induced iNOS stimulation, downregulated IL-1β and IL-6 expression at both mRNA and protein levels as well as TNF-α mRNA expression, decreased NO production and Fyn phosphorylation, reduced caspase-1 cleavage induced by moderate PrP(106-126-treatment, but had no effect on caspase-1 activation after treatment with a high concentration of PrP(106-126. Together, these results suggest that CD36 is involved in PrP(106-126-induced microglial activation and that the participation of CD36 in the interaction between PrP(106-126 and microglia may be mediated by Src tyrosine kinases. Our findings provide new insights into the mechanisms underlying the activation of microglia by neurotoxic prion peptides

Opioid-Induced Glial Activation: Mechanisms of Activation and Implications for Opioid Analgesia, Dependence, and Reward

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Mark R. Hutchinson

2007-01-01

Full Text Available This review will introduce the concept of toll-like receptor (TLR–mediated glial activation as central to all of the following: neuropathic pain, compromised acute opioid analgesia, and unwanted opioid side effects (tolerance, dependence, and reward. Attenuation of glial activation has previously been demonstrated both to alleviate exaggerated pain states induced by experimental pain models and to reduce the development of opioid tolerance. Here we demonstrate that selective acute antagonism of TLR4 results in reversal of neuropathic pain as well as potentiation of opioid analgesia. Attenuating central nervous system glial activation was also found to reduce the development of opioid dependence, and opioid reward at a behavioral (conditioned place preference and neurochemical (nucleus accumbens microdialysis of morphine-induced elevations in dopamine level of analysis. Moreover, a novel antagonism of TLR4 by (+- and (˗-isomer opioid antagonists has now been characterized, and both antiallodynic and morphine analgesia potentiating activity shown. Opioid agonists were found to also possess TLR4 agonistic activity, predictive of glial activation. Targeting glial activation is a novel and as yet clinically unexploited method for treatment of neuropathic pain. Moreover, these data indicate that attenuation of glial activation, by general or selective TLR antagonistic mechanisms, may also be a clinical method for separating the beneficial (analgesia and unwanted (tolerance, dependence, and reward actions of opioids, thereby improving the safety and efficacy of their use.

Oscillatory brain activity in spontaneous and induced sleep stages in flies.

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Yap, Melvyn H W; Grabowska, Martyna J; Rohrscheib, Chelsie; Jeans, Rhiannon; Troup, Michael; Paulk, Angelique C; van Alphen, Bart; Shaw, Paul J; van Swinderen, Bruno

2017-11-28

Sleep is a dynamic process comprising multiple stages, each associated with distinct electrophysiological properties and potentially serving different functions. While these phenomena are well described in vertebrates, it is unclear if invertebrates have distinct sleep stages. We perform local field potential (LFP) recordings on flies spontaneously sleeping, and compare their brain activity to flies induced to sleep using either genetic activation of sleep-promoting circuitry or the GABA A agonist Gaboxadol. We find a transitional sleep stage associated with a 7-10 Hz oscillation in the central brain during spontaneous sleep. Oscillatory activity is also evident when we acutely activate sleep-promoting neurons in the dorsal fan-shaped body (dFB) of Drosophila. In contrast, sleep following Gaboxadol exposure is characterized by low-amplitude LFPs, during which dFB-induced effects are suppressed. Sleep in flies thus appears to involve at least two distinct stages: increased oscillatory activity, particularly during sleep induction, followed by desynchronized or decreased brain activity.

The hydroxyflavone, fisetin, suppresses mast cell activation induced by interaction with activated T cell membranes

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Nagai, K; Takahashi, Y; Mikami, I; Fukusima, T; Oike, H; Kobori, M

2009-01-01

Background and purpose: Cell-to-cell interactions between mast cells and activated T cells are increasingly recognized as a possible mechanism in the aetiology of allergic or non-allergic inflammatory disorders. To determine the anti-allergic effect of fisetin, we examined the ability of fisetin to suppress activation of the human mast cell line, HMC-1, induced by activated Jurkat T cell membranes. Experimental approach: HMC-1 cells were incubated with or without fisetin for 15 min and then co-cultured with Jurkat T cell membranes activated by phorbol-12-myristate 13-acetate for 16 h. We determined gene expression in activated HMC-1 cells by DNA microarray and quantitative reverse transcription (RT)-PCR analysis. We also examined activation of the transcription factor NF-κB and MAP kinases (MAPKs) in activated HMC-1 cells. Key results: Fisetin suppresses cell spreading and gene expression in HMC-1 cells stimulated by activated T cell membranes. Additionally, we show that these stimulated HMC-1 cells expressed granzyme B. The stimulatory interaction also induced activation of NF-κB and MAPKs; these activations were suppressed by fisetin. Fisetin also reduced the amount of cell surface antigen CD40 and intercellular adhesion molecule-1 (ICAM-1) on activated HMC-1 cells. Conclusions and implications: Fisetin suppressed activation of HMC-1 cells by activated T cell membranes by interfering with cell-to-cell interaction and inhibiting the activity of NF-κB and MAPKs and thereby suppressing gene expression. Fisetin may protect against the progression of inflammatory diseases by limiting interactions between mast cells and activated T cells. PMID:19702784

Glucose-induced serum- and glucocorticoid-regulated kinase activation in oncofetal fibronectin expression

International Nuclear Information System (INIS)

Khan, Zia A.; Barbin, Yousef P.; Farhangkhoee, Hana; Beier, Norbert; Scholz, Wolfgang; Chakrabarti, Subrata

2005-01-01

Preferential expression of oncofetal extra domain-B fibronectin (EDB + FN), a proposed angiogenic marker, has been shown in proliferative diabetic retinopathy. High levels of glucose also increase EDB + FN expression in endothelial cells (ECs) via transforming growth factor-β1 (TGF-β1) and endothelin-1 (ET-1). The present study was aimed at elucidating the role of serum- and glucocorticoid-regulated kinase (SGK-1) in glucose-induced EDB + FN expression. Using human macro- and microvascular ECs, we show that high levels of glucose, TGF-β1, and ET-1 increase the EDB + FN expression via SGK-1 alteration at the mRNA, protein, and activity levels. Inhibition of TGF-β1 and ET-1 prevented glucose-induced SGK-1 activation and the EDB + FN expression. Furthermore, using siRNA-mediated SGK-1 gene silencing, we show that glucose-induced EDB + FN expression can be completely prevented. These findings provide first evidence of glucose-induced SGK-1 activation in altered EDB + FN expression and provide novel avenues for therapeutic modalities

Abnormal-induced theta activity supports early directed-attention network deficits in progressive MCI.

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Deiber, Marie-Pierre; Ibañez, Vicente; Missonnier, Pascal; Herrmann, François; Fazio-Costa, Lara; Gold, Gabriel; Giannakopoulos, Panteleimon

2009-09-01

The electroencephalography (EEG) theta frequency band reacts to memory and selective attention paradigms. Global theta oscillatory activity includes a posterior phase-locked component related to stimulus processing and a frontal-induced component modulated by directed attention. To investigate the presence of early deficits in the directed attention-related network in elderly individuals with mild cognitive impairment (MCI), time-frequency analysis at baseline was used to assess global and induced theta oscillatory activity (4-6Hz) during n-back working memory tasks in 29 individuals with MCI and 24 elderly controls (EC). At 1-year follow-up, 13 MCI patients were still stable and 16 had progressed. Baseline task performance was similar in stable and progressive MCI cases. Induced theta activity at baseline was significantly reduced in progressive MCI as compared to EC and stable MCI in all n-back tasks, which were similar in terms of directed attention requirements. While performance is maintained, the decrease of induced theta activity suggests early deficits in the directed-attention network in progressive MCI, whereas this network is functionally preserved in stable MCI.

Multicenter Phase II study of FOLFOX or biweekly XELOX and Erbitux (cetuximab) as first-line therapy in patients with wild-type KRAS/BRAF metastatic colorectal cancer: The FLEET study

International Nuclear Information System (INIS)

Soda, Hitoshi; Maeda, Hiromichi; Hasegawa, Junichi; Takahashi, Takao; Hazama, Shoichi; Fukunaga, Mutsumi; Kono, Emiko; Kotaka, Masahito; Sakamoto, Junichi; Nagata, Naoki; Oba, Koji; Mishima, Hideyuki

2015-01-01

The clinical benefit of cetuximab combined with oxaliplatin-based chemotherapy remains under debate. The aim of the present multicenter open-label Phase II study was to explore the efficacy and safety of biweekly administration of cetuximab and mFOLFOX-6 or XELOX as first-line chemotherapy in patients with metastatic colorectal cancer. Sixty-two patients with previously untreated KRAS/BRAF wild-type metastatic colorectal cancer were recruited to the study between April 2010 and May 2011. Patients received one of two treatment regimens, either cetuximab plus mFOLFOX-6 (FOLFOX + Cmab) or cetuximab plus biweekly XELOX (XELOX + Cmab), according to their own preference. Treatment was continued until disease progression or the appearance of intolerable toxicities. The primary endpoint was response rate; secondary endpoints were progression-free survival, overall survival, disease control rate, dose intensity, conversion rate to surgical resection, and safety. The response rates in the FOLFOX + Cmab (n = 37) and XELOX + Cmab (n = 25) groups were 64.9 % (24/37) and 72.0 % (18/25), respectively. The median PFS in the FOLFOX + Cmab and XELOX + Cmab groups was 13.1 months (95 % confidence interval [CI] 12.1–17.5) and 13.4 months (95 % CI 10.1–17.9), respectively. Neutropenia was the most frequent grade 3/4 adverse event in both groups (33.9 %), followed by anorexia, acneiform eruption, skin fissure and paronychia. A waterfall plot of tumor diameter showed prominent shrinkage of the tumors in 88.7 % of patients. The results of the present study indicate that biweekly cetuximab plus mFOLFOX-6/XELOX is an effective and tolerable treatment regimen. Biweekly administration of cetuximab requires only one hospital visit every 2 weeks, and may become a convenient treatment option for patients with KRAS/BRAF wild-type metastatic colorectal cancer

Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

International Nuclear Information System (INIS)

Wang, Bing; Wang, Xin-bao; Chen, Li-yu; Huang, Ling; Dong, Rui-zen

2013-01-01

Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancer cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells

Belinostat-induced apoptosis and growth inhibition in pancreatic cancer cells involve activation of TAK1-AMPK signaling axis

Energy Technology Data Exchange (ETDEWEB)

Wang, Bing, E-mail: wangbin69@yahoo.com; Wang, Xin-bao; Chen, Li-yu; Huang, Ling; Dong, Rui-zen

2013-07-19

Highlights: •Belinostat activates AMPK in cultured pancreatic cancer cells. •Activation of AMPK is important for belinostat-induced cytotoxic effects. •ROS and TAK1 are involved in belinostat-induced AMPK activation. •AMPK activation mediates mTOR inhibition by belinostat. -- Abstract: Pancreatic cancer accounts for more than 250,000 deaths worldwide each year. Recent studies have shown that belinostat, a novel pan histone deacetylases inhibitor (HDACi) induces apoptosis and growth inhibition in pancreatic cancer cells. However, the underlying mechanisms are not fully understood. In the current study, we found that AMP-activated protein kinase (AMPK) activation was required for belinostat-induced apoptosis and anti-proliferation in PANC-1 pancreatic cancer cells. A significant AMPK activation was induced by belinostat in PANC-1 cells. Inhibition of AMPK by RNAi knockdown or dominant negative (DN) mutation significantly inhibited belinostat-induced apoptosis in PANC-1 cells. Reversely, AMPK activator AICAR and A-769662 exerted strong cytotoxicity in PANC-1 cells. Belinostat promoted reactive oxygen species (ROS) production in PANC-1 cells, increased ROS induced transforming growth factor-β-activating kinase 1 (TAK1)/AMPK association to activate AMPK. Meanwhile, anti-oxidants N-Acetyl-Cysteine (NAC) and MnTBAP as well as TAK1 shRNA knockdown suppressed belinostat-induced AMPK activation and PANC-1 cell apoptosis. In conclusion, we propose that belinostat-induced apoptosis and growth inhibition require the activation of ROS-TAK1-AMPK signaling axis in cultured pancreatic cancer cells.

Aberrantly methylated genes in human papillary thyroid cancer and their association with BRAF/RAS mutation.

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Yasuko eKikuchi

2013-12-01

Full Text Available Cancer arises through accumulation of epigenetic and genetic alteration. Aberrant promoter methylation is a common epigenetic mechanism of gene silencing in cancer cells. We here performed genome-wide analysis of DNA methylation of promoter regions by Infinium HumanMethylation27 BeadChip, using 14 clinical papillary thyroid cancer samples and 10 normal thyroid samples. Among the 14 papillary cancer cases, 11 showed frequent aberrant methylation, but the other three cases showed no aberrant methylation at all. Distribution of the hypermethylation among cancer samples was non-random, which implied existence of a subset of preferentially methylated papillary thyroid cancer. Among 25 frequently methylated genes, methylation status of six genes (HIST1H3J, POU4F2, SHOX2, PHKG2, TLX3, HOXA7 was validated quantitatively by pyrosequencing. Epigenetic silencing of these genes in methylated papillary thyroid cancer cell lines was confirmed by gene re-expression following treatment with 5-aza-2'-deoxycytidine and trichostatin A, and detected by real-time RT-PCR. Methylation of these six genes was validated by analysis of additional 20 papillary thyroid cancer and 10 normal samples. Among the 34 cancer samples in total, 26 cancer samples with preferential methylation were significantly associated with mutation of BRAF/RAS oncogene (P=0.04, Fisher’s exact test. Thus we identified new genes with frequent epigenetic hypermethylation in papillary thyroid cancer, two subsets of either preferentially methylated or hardly methylated papillary thyroid cancer, with a concomitant occurrence of oncogene mutation and gene methylation. These hypermethylated genes may constitute potential biomarkers for papillary thyroid cancer.

Lung vagal afferent activity in rats with bleomycin-induced lung fibrosis.

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Schelegle, E S; Walby, W F; Mansoor, J K; Chen, A T

2001-05-01

Bleomycin treatment in rats results in pulmonary fibrosis that is characterized by a rapid shallow breathing pattern, a decrease in quasi-static lung compliance and a blunting of the Hering-Breuer Inflation Reflex. We examined the impulse activity of pulmonary vagal afferents in anesthetized, mechanically ventilated rats with bleomycin-induced lung fibrosis during the ventilator cycle and static lung inflations/deflations and following the injection of capsaicin into the right atrium. Bleomycin enhanced volume sensitivity of slowly adapting stretch receptors (SARs), while it blunted the sensitivity of these receptors to increasing transpulmonary pressure. Bleomycin treatment increased the inspiratory activity, while it decreased the expiratory activity of rapidly adapting stretch receptors (RARs). Pulmonary C-fiber impulse activity did not appear to be affected by bleomycin treatment. We conclude that the fibrosis-related shift in discharge profile and enhanced volume sensitivity of SARs combined with the increased inspiratory activity of RARs contributes to the observed rapid shallow breathing of bleomycin-induced lung fibrosis.

Anesthetic propofol attenuates the isoflurane-induced caspase-3 activation and Aβ oligomerization.

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Yiying Zhang

Full Text Available Accumulation and deposition of β-amyloid protein (Aβ are the hallmark features of Alzheimer's disease. The inhalation anesthetic isoflurane has been shown to induce caspase activation and increase Aβ accumulation. In addition, recent studies suggest that isoflurane may directly promote the formation of cytotoxic soluble Aβ oligomers, which are thought to be the key pathological species in AD. In contrast, propofol, the most commonly used intravenous anesthetic, has been reported to have neuroprotective effects. We therefore set out to compare the effects of isoflurane and propofol alone and in combination on caspase-3 activation and Aβ oligomerization in vitro and in vivo. Naïve and stably-transfected H4 human neuroglioma cells that express human amyloid precursor protein, the precursor for Aβ; neonatal mice; and conditioned cell culture media containing secreted human Aβ40 or Aβ42 were treated with isoflurane and/or propofol. Here we show for the first time that propofol can attenuate isoflurane-induced caspase-3 activation in cultured cells and in the brain tissues of neonatal mice. Furthermore, propofol-mediated caspase inhibition occurred when there were elevated levels of Aβ. Finally, isoflurane alone induces Aβ42, but not Aβ40, oligomerization, and propofol can inhibit the isoflurane-mediated oligomerization of Aβ42. These data suggest that propofol may mitigate the caspase-3 activation by attenuating the isoflurane-induced Aβ42 oligomerization. Our findings provide novel insights into the possible mechanisms of isoflurane-induced neurotoxicity that may aid in the development of strategies to minimize potential adverse effects associated with the administration of anesthetics to patients.

Allergen-Removed Rhus verniciflua Extract Induces Ovarian Cancer Cell Death via JNK Activation.

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Kang, Se-Hui; Hwang, In-Hu; Son, Eunju; Cho, Chong-Kwan; Choi, Jong-Soon; Park, Soo-Jung; Jang, Byeong-Churl; Lee, Kyung-Bok; Lee, Zee-Won; Lee, Jong Hoon; Yoo, Hwa-Seung; Jang, Ik-Soon

2016-01-01

Nuclear factor-[Formula: see text]B (NF-[Formula: see text]B)/Rel transcription factors are best known for their central roles in promoting cell survival in cancer. NF-[Formula: see text]B antagonizes tumor necrosis factor (TNF)-[Formula: see text]-induced apoptosis through a process involving attenuation of the c-Jun-N-terminal kinase (JNK). However, the role of JNK activation in apoptosis induced by negative regulation of NF-[Formula: see text]B is not completely understood. We found that allergen-removed Rhus verniciflua Stokes (aRVS) extract-mediated NF-[Formula: see text]B inhibition induces apoptosis in SKOV-3 ovarian cancer cells via the serial activation of caspases and SKOV-3 cells are most specifically suppressed by aRVS. Here, we show that in addition to activating caspases, aRVS extract negatively modulates the TNF-[Formula: see text]-mediated I[Formula: see text]B/NF-[Formula: see text]B pathway to promote JNK activation, which results in apoptosis. When the cytokine TNF-[Formula: see text] binds to the TNF receptor, I[Formula: see text]B dissociates from NF-[Formula: see text]B. As a result, the active NF-[Formula: see text]B translocates to the nucleus. aRVS extract (0.5[Formula: see text]mg/ml) clearly prevented NF-[Formula: see text]B from mobilizing to the nucleus, resulting in the upregulation of JNK phosphorylation. This subsequently increased Bax activation, leading to marked aRVS-induced apoptosis, whereas the JNK inhibitor SP600125 in aRVS extract treated SKOV-3 cells strongly inhibited Bax. Bax subfamily proteins induced apoptosis through caspase-3. Thus, these results indicate that aRVS extract contains components that inhibit NF-[Formula: see text]B signaling to upregulate JNK activation in ovarian cancer cells and support the potential of aRVS as a therapeutic agent for ovarian cancer.

Arctigenin Inhibits Adipogenesis by Inducing AMPK Activation and Reduces Weight Gain in High-Fat Diet-Induced Obese Mice.

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Han, Yo-Han; Kee, Ji-Ye; Park, Jinbong; Kim, Hye-Lin; Jeong, Mi-Young; Kim, Dae-Seung; Jeon, Yong-Deok; Jung, Yunu; Youn, Dong-Hyun; Kang, JongWook; So, Hong-Seob; Park, Raekil; Lee, Jong-Hyun; Shin, Soyoung; Kim, Su-Jin; Um, Jae-Young; Hong, Seung-Heon

2016-09-01

Although arctigenin (ARC) has been reported to have some pharmacological effects such as anti-inflammation, anti-cancer, and antioxidant, there have been no reports on the anti-obesity effect of ARC. The aim of this study is to investigate whether ARC has an anti-obesity effect and mediates the AMP-activated protein kinase (AMPK) pathway. We investigated the anti-adipogenic effect of ARC using 3T3-L1 pre-adipocytes and human adipose tissue-derived mesenchymal stem cells (hAMSCs). In high-fat diet (HFD)-induced obese mice, whether ARC can inhibit weight gain was investigated. We found that ARC reduced weight gain, fat pad weight, and triglycerides in HFD-induced obese mice. ARC also inhibited the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα) in in vitro and in vivo. Furthermore, ARC induced the AMPK activation resulting in down-modulation of adipogenesis-related factors including PPARγ, C/EBPα, fatty acid synthase, adipocyte fatty acid-binding protein, and lipoprotein lipase. This study demonstrates that ARC can reduce key adipogenic factors by activating the AMPK in vitro and in vivo and suggests a therapeutic implication of ARC for obesity treatment. J. Cell. Biochem. 117: 2067-2077, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

TATA-binding protein-associated factor 7 regulates polyamine transport activity and polyamine analog-induced apoptosis.

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Fukuchi, Junichi; Hiipakka, Richard A; Kokontis, John M; Nishimura, Kazuhiro; Igarashi, Kazuei; Liao, Shutsung

2004-07-16

Identification of the polyamine transporter gene will be useful for modulating polyamine accumulation in cells and should be a good target for controlling cell proliferation. Polyamine transport activity in mammalian cells is critical for accumulation of the polyamine analog methylglyoxal bis(guanylhydrazone) (MGBG) that induces apoptosis, although a gene responsible for transport activity has not been identified. Using a retroviral gene trap screen, we generated MGBG-resistant Chinese hamster ovary (CHO) cells to identify genes involved in polyamine transport activity. One gene identified by the method encodes TATA-binding protein-associated factor 7 (TAF7), which functions not only as one of the TAFs, but also a coactivator for c-Jun. TAF7-deficient cells had decreased capacity for polyamine uptake (20% of CHO cells), decreased AP-1 activation, as well as resistance to MGBG-induced apoptosis. Stable expression of TAF7 in TAF7-deficient cells restored transport activity (55% of CHO cells), AP-1 gene transactivation (100% of CHO cells), and sensitivity to MGBG-induced apoptosis. Overexpression of TAF7 in CHO cells did not increase transport activity, suggesting that TAF7 may be involved in the maintenance of basal activity. c-Jun NH2-terminal kinase inhibitors blocked MGBG-induced apoptosis without alteration of polyamine transport. Decreased TAF7 expression, by RNA interference, in androgen-independent human prostate cancer LN-CaP104-R1 cells resulted in lower polyamine transport activity (25% of control) and resistance to MGBG-induced growth arrest. Taken together, these results reveal a physiological function of TAF7 as a basal regulator for mammalian polyamine transport activity and MGBG-induced apoptosis.

A bacterial cocaine esterase protects against cocaine-induced epileptogenic activity and lethality.

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Jutkiewicz, Emily M; Baladi, Michelle G; Cooper, Ziva D; Narasimhan, Diwahar; Sunahara, Roger K; Woods, James H

2009-09-01

Cocaine toxicity results in cardiovascular complications, seizures, and death and accounts for approximately 20% of drug-related emergency department visits every year. Presently, there are no treatments to eliminate the toxic effects of cocaine. The present study hypothesizes that a bacterial cocaine esterase with high catalytic efficiency would provide rapid and robust protection from cocaine-induced convulsions, epileptogenic activity, and lethality. Cocaine-induced paroxysmal activity and convulsions were evaluated in rats surgically implanted with radiotelemetry devices (N=6 per treatment group). Cocaine esterase was administered 1 minute after a lethal dose of cocaine or after cocaine-induced convulsions to determine the ability of the enzyme to prevent or reverse, respectively, the effects of cocaine. The cocaine esterase prevented all cocaine-induced electroencephalographic changes and lethality. This effect was specific for cocaine because the esterase did not prevent convulsions and death induced by a cocaine analog, (-)-2beta-carbomethoxy-3beta-phenyltropane. The esterase prevented lethality even after cocaine-induced convulsions occurred. In contrast, the short-acting benzodiazepine, midazolam, prevented cocaine-induced convulsions but not the lethal effects of cocaine. The data showed that cocaine esterase successfully degraded circulating cocaine to prevent lethality and that cocaine-induced convulsions alone are not responsible for the lethal effects of cocaine in this model. Therefore, further investigation into the use of cocaine esterase for treating cocaine overdose and its toxic effects is warranted.

The NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation

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Shi Fushan

2012-07-01

Full Text Available Abstract Background Prion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal disease-associated prion protein, PrPSc. In prion-infected brains, activated microglia are often present in the vicinity of PrPSc aggregates, and microglial activation is thought to play a key role in the pathogenesis of prion diseases. Although interleukin (IL-1β release by prion-induced microglia has been widely reported, the mechanism by which primed microglia become activated and secrete IL-1β in prion diseases has not yet been elucidated. In this study, we investigated the role of the NACHT, LRR and PYD domains-containing protein (NALP3 inflammasome in IL-1β release from lipopolysaccharide (LPS-primed microglia after exposure to a synthetic neurotoxic prion fragment (PrP106-126. Methods The inflammasome components NALP3 and apoptosis-associated speck-like protein (ASC were knocked down by gene silencing. IL-1β production was assessed using ELISA. The mRNA expression of NALP3, ASC, and pro-inflammatory factors was measured by quantitative PCR. Western blot analysis was used to detect the protein level of NALP3, ASC, caspase-1 and nuclear factor-κB. Results We found that that PrP106-126-induced IL-1β release depends on NALP3 inflammasome activation, that inflammasome activation is required for the synthesis of pro-inflammatory and chemotactic factors by PrP106-126-activated microglia, that inhibition of NF-κB activation abrogated PrP106-126-induced NALP3 upregulation, and that potassium efflux and production of reactive oxygen species were implicated in PrP106-126-induced NALP3 inflammasome activation in microglia. Conclusions We conclude that the NALP3 inflammasome is involved in neurotoxic prion peptide-induced microglial activation. To our knowledge, this is the first time that strong evidence for the involvement of NALP3 inflammasome in prion-associated inflammation has been found.

Differential modulatory effects of GSK-3β and HDM2 on sorafenib-induced AIF nuclear translocation (programmed necrosis in melanoma

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Mier James W

2011-09-01

Full Text Available Abstract Background GSK-3β phosphorylates numerous substrates that govern cell survival. It phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent ubiquitination, and proteasomal degradation. GSK-3β can either enhance or inhibit programmed cell death, depending on the nature of the pro-apoptotic stimulus. We previously showed that the multikinase inhibitor sorafenib activated GSK-3β and that this activation attenuated the cytotoxic effects of the drug in various BRAF-mutant melanoma cell lines. In this report, we describe the results of studies exploring the effects of GSK-3β on the cytotoxicity and antitumor activity of sorafenib combined with the HDM2 antagonist MI-319. Results MI-319 alone increased p53 levels and p53-dependent gene expression in melanoma cells but did not induce programmed cell death. Its cytotoxicity, however, was augmented in some melanoma cell lines by the addition of sorafenib. In responsive cell lines, the MI-319/sorafenib combination induced the disappearance of p53 from the nucleus, the down modulation of Bcl-2 and Bcl-xL, the translocation of p53 to the mitochondria and that of AIF to the nuclei. These events were all GSK-3β-dependent in that they were blocked with a GSK-3β shRNA and facilitated in otherwise unresponsive melanoma cell lines by the introduction of a constitutively active form of the kinase (GSK-3β-S9A. These modulatory effects of GSK-3β on the activities of the sorafenib/MI-319 combination were the exact reverse of its effects on the activities of sorafenib alone, which induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells in which GSK-3β activity was either down modulated or constitutively low. In A375 xenografts, the antitumor effects of sorafenib and MI-319 were additive and associated with the down modulation of Bcl-2 and Bcl-xL, the nuclear translocation of AIF, and increased suppression of tumor angiogenesis

Activating the Wnt/β-Catenin Pathway for the Treatment of Melanoma--Application of LY2090314, a Novel Selective Inhibitor of Glycogen Synthase Kinase-3.

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Jennifer M Atkinson

Full Text Available It has previously been observed that a loss of β-catenin expression occurs with melanoma progression and that nuclear β-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/β-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3β isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized β-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10 nM after 72hr of treatment in contrast to other solid tumor cell lines (IC50 >10 uM as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that β-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by β-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.

Shanxi Aged Vinegar Protects against Alcohol-Induced Liver Injury via Activating Nrf2-Mediated Antioxidant and Inhibiting TLR4-Induced Inflammatory Response

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Ting Xia

2018-06-01

Full Text Available Shanxi aged vinegar (SAV is a typical fermented and antioxidant food, which has various health-promoting effects. This work aimed to explore the effects of SAV on alcohol-induced liver injury. A mice model of alcoholic liver injury was established to illuminate its potential mechanisms. All mice pretreated with SAV and then received an ethanol solution (50% w/v, 4.8 g/kg b.w.. The results showed that SAV ameliorated alcohol-induced histological changes and elevation of liver enzymes. SAV attenuated alcohol-induced oxidative stress by declining levels of hepatic oxidants, and restoring depletion of antioxidant enzyme activities in mice livers. Moreover, SAV alleviated alcohol-induced oxidative damage by activating nuclear factor erythroid-2-related factor 2 (Nrf2-mediated signal pathway. In addition, SAV prevented alcohol-induced inflammation by suppressing lipopolysaccharide (LPS level and activities of pro-inflammatory enzymes, and regulating inflammatory cytokines. SAV inhibited alcohol-induced inflammation through down-regulating the expression of Toll-like receptor 4 (TLR4-mediated inflammatory response. The findings provide crucial evidence for elucidating the hepatoprotective mechanisms of SAV and encourage the future application of SAV as a functional food for liver protection.

Structures of the inactive and active states of RIP2 kinase inform on the mechanism of activation.

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Erika Pellegrini

Full Text Available Innate immune receptors NOD1 and NOD2 are activated by bacterial peptidoglycans leading to recruitment of adaptor kinase RIP2, which, upon phosphorylation and ubiquitination, becomes a scaffold for downstream effectors. The kinase domain (RIP2K is a pharmaceutical target for inflammatory diseases caused by aberrant NOD2-RIP2 signalling. Although structures of active RIP2K in complex with inhibitors have been reported, the mechanism of RIP2K activation remains to be elucidated. Here we analyse RIP2K activation by combining crystal structures of the active and inactive states with mass spectrometric characterization of their phosphorylation profiles. The active state has Helix αC inwardly displaced and the phosphorylated Activation Segment (AS disordered, whilst in the inactive state Helix αC is outwardly displaced and packed against the helical, non-phosphorylated AS. Biophysical measurements show that the active state is a stable dimer whilst the inactive kinase is in a monomer-dimer equilibrium, consistent with the observed structural differences at the dimer interface. We conclude that RIP2 kinase auto-phosphorylation is intimately coupled to dimerization, similar to the case of BRAF. Our results will help drug design efforts targeting RIP2 as a potential treatment for NOD2-RIP2 related inflammatory diseases.

PIK3CA activating mutation in colorectal carcinoma: associations with molecular features and survival.

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Christophe Rosty

Full Text Available Mutations in PIK3CA are present in 10 to 15% of colorectal carcinomas. We aimed to examine how PIK3CA mutations relate to other molecular alterations in colorectal carcinoma, to pathologic phenotype and survival. PIK3CA mutation testing was carried out using direct sequencing on 757 incident tumors from the Melbourne Collaborative Cohort Study. The status of O-6-methylguanine-DNA methyltransferase (MGMT was assessed using both immunohistochemistry and methyLight techniques. Microsatellite instability, CpG island phenotype (CIMP, KRAS and BRAF V600E mutation status, and pathology review features were derived from previous reports. PIK3CA mutation was observed in 105 of 757 (14% of carcinomas, characterized by location in the proximal colon (54% vs. 34%; P<0.001 and an increased frequency of KRAS mutation (48% vs. 25%; P<0.001. High-levels of CIMP were more frequently found in PIK3CA-mutated tumors compared with PIK3CA wild-type tumors (22% vs. 11%; P = 0.004. There was no difference in the prevalence of BRAF V600E mutation between these two tumor groups. PIK3CA-mutated tumors were associated with loss of MGMT expression (35% vs. 20%; P = 0.001 and the presence of tumor mucinous differentiation (54% vs. 32%; P<0.001. In patients with wild-type BRAF tumors, PIK3CA mutation was associated with poor survival (HR 1.51 95% CI 1.04-2.19, P = 0.03. In summary, PIK3CA-mutated colorectal carcinomas are more likely to develop in the proximal colon, to demonstrate high levels of CIMP, KRAS mutation and loss of MGMT expression. PIK3CA mutation also contributes to significantly decreased survival for patients with wild-type BRAF tumors.

Non-transactivational, dual pathways for LPA-induced Erk1/2 activation in primary cultures of brown pre-adipocytes

International Nuclear Information System (INIS)

Holmstroem, Therese E.; Mattsson, Charlotte L.; Wang, Yanling; Iakovleva, Irina; Petrovic, Natasa; Nedergaard, Jan

2010-01-01

In many cell types, G-protein-coupled receptor (GPCR)-induced Erk1/2 MAP kinase activation is mediated via receptor tyrosine kinase (RTK) transactivation, in particular via the epidermal growth factor (EGF) receptor. Lysophosphatidic acid (LPA), acting via GPCRs, is a mitogen and MAP kinase activator in many systems, and LPA can regulate adipocyte proliferation. The mechanism by which LPA activates the Erk1/2 MAP kinase is generally accepted to be via EGF receptor transactivation. In primary cultures of brown pre-adipocytes, EGF can induce Erk1/2 activation, which is obligatory and determinant for EGF-induced proliferation of these cells. Therefore, we have here examined whether LPA, via EGF transactivation, can activate Erk1/2 in brown pre-adipocytes. We found that LPA could induce Erk1/2 activation. However, the LPA-induced Erk1/2 activation was independent of transactivation of EGF receptors (or PDGF receptors) in these cells (whereas in transformed HIB-1B brown adipocytes, the LPA-induced Erk1/2 activation indeed proceeded via EGF receptor transactivation). In the brown pre-adipocytes, LPA instead induced Erk1/2 activation via two distinct non-transactivational pathways, one G i -protein dependent, involving PKC and Src activation, the other, a PTX-insensitive pathway, involving PI3K (but not Akt) activation. Earlier studies showing LPA-induced Erk1/2 activation being fully dependent on RTK transactivation have all been performed in cell lines and transfected cells. The present study implies that in non-transformed systems, RTK transactivation may not be involved in the mediation of GPCR-induced Erk1/2 MAP kinase activation.

Hyperthyroidism enhances 5-HT-induced contraction of the rat pulmonary artery: role of calcium-activated chloride channel activation.

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Oriowo, Mabayoje A; Oommen, Elsie; Khan, Islam

2011-11-01

Experimentally-induced hyperthyroidism in rodents is associated with signs and symptoms of pulmonary hypertension. The main objective of the present study was to investigate the effect of thyroxine-induced pulmonary hypertension on the contractile response of the pulmonary artery to 5-HT and the possible underlying signaling pathway. 5-HT concentration-dependently contracted artery segments from control and thyroxine-treated rats with pD(2) values of 5.04 ± 0.19 and 5.34 ± 0.14, respectively. The maximum response was significantly greater in artery segments from thyroxine-treated rats. Neither BW 723C86 (5-HT(2B)-receptor agonist) nor CP 93129 (5-HT(1B)-receptor agonist) contracted ring segments of the pulmonary artery from control and thyroxine-treated rats at concentrations up to 10(-4)M. There was no significant difference in the level of expression of 5-HT(2A)-receptor protein between the two groups. Ketanserin (3 × 10(-8)M) produced a rightward shift of the concentration-response curve to 5-HT in both groups with equal potency (-logK(B) values were 8.1 ± 0.2 and 7.9 ± 0.1 in control and thyroxine-treated rats, respectively). Nifedipine (10(-6)M) inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. The calcium-activated chloride channel blocker, niflumic acid (10(-4)M) also inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. It was concluded that hyperthyroidism enhanced 5-HT-induced contractions of the rat pulmonary artery by a mechanism involving increased activity of calcium-activated chloride channels. Copyright © 2011 Elsevier B.V. All rights reserved.

UVC-induced apoptosis in Dubca cells is independent of JNK activation and p53Ser-15 phosphorylation

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Chathoth, Shahanas; Thayyullathil, Faisal; Hago, Abdulkader; Shahin, Allen; Patel, Mahendra; Galadari, Sehamuddin

2009-01-01

Ultraviolet C (UVC) irradiation in mammalian cell lines activates a complex signaling network that leads to apoptosis. By using Dubca cells as a model system, we report the presence of a UVC-induced apoptotic pathway that is independent of c-Jun N-terminal kinases (JNKs) activation and p53 phosphorylation at Ser 15 . Irradiation of Dubca cells with UVC results in a rapid JNK activation and phosphorylation of its downstream target c-Jun, as well as, phosphorylation of activating transcription factor 2 (ATF2). Pre-treatment with JNK inhibitor, SP600125, inhibited UVC-induced c-Jun phosphorylation without preventing UVC-induced apoptosis. Similarly, inhibition of UVC-induced p53 phosphorylation did not prevent Dubca cell apoptosis, suggesting that p53 Ser-15 phosphorylation is not associated with UVC-induced apoptosis signaling. The pan-caspase inhibitor z-VAD-fmk inhibited UVC-induced PARP cleavage, DNA fragmentation, and ultimately apoptosis of Dubca cells. Altogether, our study clearly indicates that UVC-induced apoptosis is independent of JNK and p53 activation in Dubca cells, rather, it is mediated through a caspase dependent pathway. Our findings are not in line with the ascribed critical role for JNKs activation, and downstream phosphorylation of targets such as c-Jun and ATF2 in UVC-induced apoptosis.

Impurities and evaluation of induced activity of SiCf/SiC composites

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Noda, Tetsuji; Araki, Hiroshi; Ito, Shinji; Fujita, Mitsutane; Maki, Koichi

1997-01-01

Impurity of SiC f /SiC composites prepared by CVI was analyzed by neutron activation analysis and glow discharge mass spectrometry. The evaluation of the induced activity of the composites based on the chemical compositions was made using a simulation calculation for fusion reactor blanket. Impurities of 35 elements were detected in the composites. However the total concentration of metallic impurities was below 20 mass ppm. The analyses of induced activity of the composites show that the dose rate decreases by about 5 orders of magnitude in a day after the shutdown. It is recommended that the purification of SiC fibers is necessary to reduce the activity by 10 9 after several ten years cooling of fusion reactors. (author)

p38 mitogen-activated protein kinase up-regulates NF-κB transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

International Nuclear Information System (INIS)

Ji, Guoping; Liu, Dongxu; Liu, Jing; Gao, Hui; Yuan, Xiao; Shen, Gang

2010-01-01

p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-κB activation during myogenesis, not through down-regulation of degradation of IκB-α, and consequent translocation of the p65 subunit of NF-κB to the nucleus. It is likely that stretch-induced NF-κB activation by phosphorylation of p65 NF-κB. Moreover, depletion of p38α using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-κB activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-κB signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The α isoform of p38MAP kinase regulates the transcriptional activation of NF-κB following stimulation with cyclic stretch.

p38 mitogen-activated protein kinase up-regulates NF-{kappa}B transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

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Ji, Guoping [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China); Liu, Dongxu [Department of Orthodontics, College of Stomatology, Shandong University, Jinan, Shandong Province 250012 (China); Liu, Jing [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Gao, Hui [Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041 (China); Yuan, Xiao, E-mail: yuanxiaoqd@163.com [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Shen, Gang, E-mail: ganshen2007@163.com [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China)

2010-01-01

p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-{kappa}B in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-{kappa}B activation during myogenesis, not through down-regulation of degradation of I{kappa}B-{alpha}, and consequent translocation of the p65 subunit of NF-{kappa}B to the nucleus. It is likely that stretch-induced NF-{kappa}B activation by phosphorylation of p65 NF-{kappa}B. Moreover, depletion of p38{alpha} using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-{kappa}B activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-{kappa}B signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The {alpha} isoform of p38MAP kinase regulates the transcriptional activation of NF-{kappa}B following stimulation with cyclic stretch.

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

DEFF Research Database (Denmark)

Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

2013-01-01

knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

Irradiation-induced telomerase activity and gastric cancer risk: a case-control analysis in a Chinese Han population

International Nuclear Information System (INIS)

He, Xianli; Qiao, Qing; Ge, Naijian; Nan, Jing; Shen, Shuqun; Wang, Zizhong; Yang, Yefa; Bao, Guoqiang

2010-01-01

Telomerase expression is one of the characteristics of gastric cancer (GC) cells and telomerase activity is frequently up-regulated by a variety of mechanisms during GC development. Therefore, we hypothesized that elevated levels of activated telomerase might enhance GC risk due to increased propagation of cells with DNA damage, such as induced by γ-radiation. To explore this hypothesis, 246 GC cases and 246 matched controls were recruited in our case-control study. TRAP-ELISA was used to assess the levels of telomerase activity at baseline and after γ-radiation and the γ-radiation-induced telomerase activity (defined as after γ-irradiation/baseline) in cultured peripheral blood lymphocytes (PBLs). Our data showed that there was no significant difference for the baseline telomerase activity between GC cases and controls (10.17 ± 7.21 vs. 11.02 ± 8.03, p = 0.168). However, after γ-radiation treatment, γ-radiation-induced telomerase activity was significantly higher in the cases than in the controls (1.51 ± 0.93 vs. 1.22 ± 0.66, p < 0.001). Using the median value of γ-radiation-induced telomerase activity in the controls as a cutoff point, we observed that high γ-radiation-induced telomerase activity was associated with a significantly increased GC risk (adjusted odds ratio, 2.45; 95% confidence interval, 1.83-3.18). Moreover, a dose response association was noted between γ-radiation-induced telomerase activity and GC risk. Age, but not sex, smoking and drinking status seem to have a modulating effect on the γ-radiation-induced telomerase activities in both cases and controls. Overall, our findings for the first time suggest that the increased γ-radiation-induced telomerase activity in PBLs might be associated with elevated GC risk. Further confirmation of this association using a prospective study design is warranted

Clinical responses to ERK inhibition in BRAFV600E-mutant colorectal cancer predicted using a computational model.

Science.gov (United States)

Kirouac, Daniel C; Schaefer, Gabriele; Chan, Jocelyn; Merchant, Mark; Orr, Christine; Huang, Shih-Min A; Moffat, John; Liu, Lichuan; Gadkar, Kapil; Ramanujan, Saroja

2017-01-01

Approximately 10% of colorectal cancers harbor BRAF V600E mutations, which constitutively activate the MAPK signaling pathway. We sought to determine whether ERK inhibitor (GDC-0994)-containing regimens may be of clinical benefit to these patients based on data from in vitro (cell line) and in vivo (cell- and patient-derived xenograft) studies of cetuximab (EGFR), vemurafenib (BRAF), cobimetinib (MEK), and GDC-0994 (ERK) combinations. Preclinical data was used to develop a mechanism-based computational model linking cell surface receptor (EGFR) activation, the MAPK signaling pathway, and tumor growth. Clinical predictions of anti-tumor activity were enabled by the use of tumor response data from three Phase 1 clinical trials testing combinations of EGFR, BRAF, and MEK inhibitors. Simulated responses to GDC-0994 monotherapy (overall response rate = 17%) accurately predicted results from a Phase 1 clinical trial regarding the number of responding patients (2/18) and the distribution of tumor size changes ("waterfall plot"). Prospective simulations were then used to evaluate potential drug combinations and predictive biomarkers for increasing responsiveness to MEK/ERK inhibitors in these patients.

VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

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Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

2010-01-01

Research highlights: → Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. → CoCl 2 -induced VEGF secretion in mast cells occurs by a Ca 2+ -insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. → Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits FcεRI-dependent anaphylactic degranulation in mast cells. → Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl 2 ) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl 2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl 2 -induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl 2 -induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl 2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

Energy Technology Data Exchange (ETDEWEB)

Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

2010-10-15

Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells.

Directory of Open Access Journals (Sweden)

Qinghe Chen

2010-12-01

Full Text Available Resveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol.Resveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by

Oxidized LDL Induces Alternative Macrophage Phenotype through Activation of CD36 and PAFR

Directory of Open Access Journals (Sweden)

Francisco J. Rios

2013-01-01

Full Text Available OxLDL is recognized by macrophage scavenger receptors, including CD36; we have recently found that Platelet-Activating Factor Receptor (PAFR is also involved. Since PAFR in macrophages is associated with suppressor function, we examined the effect of oxLDL on macrophage phenotype. It was found that the presence of oxLDL during macrophage differentiation induced high mRNA levels to IL-10, mannose receptor, PPARγ and arginase-1 and low levels of IL-12 and iNOS. When human THP-1 macrophages were pre-treated with oxLDL then stimulated with LPS, the production of IL-10 and TGF-β significantly increased, whereas that of IL-6 and IL-8 decreased. In murine TG-elicited macrophages, this protocol significantly reduced NO, iNOS and COX2 expression. Thus, oxLDL induced macrophage differentiation and activation towards the alternatively activated M2-phenotype. In murine macrophages, oxLDL induced TGF-β, arginase-1 and IL-10 mRNA expression, which were significantly reduced by pre-treatment with PAFR antagonists (WEB and CV or with antibodies to CD36. The mRNA expression of IL-12, RANTES and CXCL2 were not affected. We showed that this profile of macrophage activation is dependent on the engagement of both CD36 and PAFR. We conclude that oxLDL induces alternative macrophage activation by mechanisms involving CD36 and PAFR.

Cisplatin-induced Casepase-3 activation in different tumor cells

Science.gov (United States)

Shi, Hua; Li, Xiao; Su, Ting; Zhang, Yu-Hai

2008-12-01

Apoptosis plays an essential role in normal organism development which is one of the main types of programmed cell death to help tissues maintain homeostasis. Defective apoptosis can result in cell accumulation and therefore effects on tumor pathogenesis, progression and therapy resistance. A family of proteins, known as caspases, is typically activated in the early stages of apoptosis. Therefore, studying the kinetics of activation of caspases induced by antitumor drugs can contribute to antitumor drug discovery and explanation of the molecular mechanisms. This paper detected the Caspase-3 activity induced by cisplatin in human adenoid cystic carcinoma cell line (ACC-M), human hepatocellular liver carcinoma cell line (HepG2) and human epithelial carcinoma cell line (Hela) with stably expressing ECFP-DEVDDsRed (CD3) probe, a fluorescent probe consisting of Enhanced Cyan Fluorescent Protein (ECFP), red fluorescent protein (DsRed) and a linker with a recognition site of Caspase-3, by using the capillary electrophoresis (CE) and fluorescence resonance energy transfer (FRET) imaging system. Under the same concentration of cisplatin, ACC-M cells responded the most rapidly, and then HepG2 cells and Hela cells, respectively, in the early 30 hours. Later, HepG2 cells represented acceleration in the Caspase-3 activation speed and reached full activation the earliest comparing to other two cell types. The results demonstrated that ACC-M cell is more sensitive than the other two cell types under the treatment of cisplatin.

Macroscopic tunneling, decoherence and noise-induced activation

Energy Technology Data Exchange (ETDEWEB)

Lombardo, Fernando C; Monteoliva, Diana; Villar, Paula I [Departamento de Fisica Juan Jose Giambiagi, Facultad de Ciencias Exactas y Naturales, UBA, Ciudad Universitaria, Pabellon I, 1428 Buenos Aires (Argentina)

2007-05-15

We study the effects of the environment at zero temperature on tunneling in an open system described by a static double-well potential. We show that the evolution of the system in an initial Schroedinger cat state, can be summarized in terms of three main physical phenomena, namely decoherence, quantum tunneling and noise-induced activation. Using large-scale numerical simulations, we obtain a detailed picture of the main stages of the evolution and of the relevant dynamical processes.

Statins activate GATA-6 and induce differentiated vascular smooth muscle cells

International Nuclear Information System (INIS)

Wada, Hiromichi; Abe, Mitsuru; Ono, Koh; Morimoto, Tatsuya; Kawamura, Teruhisa; Takaya, Tomohide; Satoh, Noriko; Fujita, Masatoshi; Kita, Toru; Shimatsu, Akira; Hasegawa, Koji

2008-01-01

The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 μM of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-α-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 μM), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs

Statins activate GATA-6 and induce differentiated vascular smooth muscle cells

Energy Technology Data Exchange (ETDEWEB)

Wada, Hiromichi [Division of Translational Research, National Hospital Organization Kyoto Medical Center, 1-1 Mukaihata-cho, Fukakusa, Fushimi-ku, Kyoto 612-8555 (Japan); Abe, Mitsuru; Ono, Koh [Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto (Japan); Morimoto, Tatsuya; Kawamura, Teruhisa; Takaya, Tomohide [Division of Translational Research, National Hospital Organization Kyoto Medical Center, 1-1 Mukaihata-cho, Fukakusa, Fushimi-ku, Kyoto 612-8555 (Japan); Satoh, Noriko [Division of Metabolic Research, National Hospital Organization Kyoto Medical Center, Kyoto (Japan); Fujita, Masatoshi [Department of Human Health Sciences, Graduate School of Medicine, Kyoto University, Kyoto (Japan); Kita, Toru [Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto (Japan); Shimatsu, Akira [Clinical Research Institute, National Hospital Organization Kyoto Medical Center, Kyoto (Japan); Hasegawa, Koji [Division of Translational Research, National Hospital Organization Kyoto Medical Center, 1-1 Mukaihata-cho, Fukakusa, Fushimi-ku, Kyoto 612-8555 (Japan)

2008-10-03

The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) beyond cholesterol lowering involve their direct actions on vascular smooth muscle cells (VSMCs). However, the effects of statins on phenotypic modulation of VSMCs are unknown. We herein show that simvastatin (Sm) and atorvastatin (At) inhibited DNA synthesis in human aortic VSMCs dose-dependently, while cell toxicity was not observed below the concentration of 1 {mu}M of Sm or 100 nM of At. Stimulating proliferative VSMCs with Sm or At induced the expression of SM-{alpha}-actin and SM-MHC, highly specific markers of differentiated phenotype. Sm up-regulated the binding activity of GATA-6 to SM-MHC GATA site and activated the transfected SM-MHC promoter in proliferative VSMCs, while mutating the GATA-6 binding site abolished this activation. Geranylgeranylpyrophosphate (10 {mu}M), an inhibitor of Rho family proteins, abolished the statin-mediated induction of the differentiated phenotype in VSMCs. These findings suggest that statins activate GATA-6 and induce differentiated VSMCs.

Activation of PPARα by Wy-14643 ameliorates systemic lipopolysaccharide-induced acute lung injury

International Nuclear Information System (INIS)

Yoo, Seong Ho; Abdelmegeed, Mohamed A.; Song, Byoung-Joon

2013-01-01

Highlights: •Activation of PPARα attenuated LPS-mediated acute lung injury. •Pretreatment with Wy-14643 decreased the levels of IFN-γ and IL-6 in ALI. •Nitrosative stress and lipid peroxidation were downregulated by PPARα activation. •PPARα agonists may be potential therapeutic targets for acute lung injury. -- Abstract: Acute lung injury (ALI) is a major cause of mortality and morbidity worldwide. The activation of peroxisome proliferator-activated receptor-α (PPARα) by its ligands, which include Wy-14643, has been implicated as a potential anti-inflammatory therapy. To address the beneficial efficacy of Wy-14643 for ALI along with systemic inflammation, the in vivo role of PPARα activation was investigated in a mouse model of lipopolysaccharide (LPS)-induced ALI. Using age-matched Ppara-null and wild-type mice, we demonstrate that the activation of PPARα by Wy-14643 attenuated LPS-mediated ALI. This was evidenced histologically by the significant alleviation of inflammatory manifestations and apoptosis observed in the lung tissues of wild-type mice, but not in the corresponding Ppara-null mice. This protective effect probably resulted from the inhibition of LPS-induced increases in pro-inflammatory cytokines and nitroxidative stress levels. These results suggest that the pharmacological activation of PPARα might have a therapeutic effect on LPS-induced ALI

Activation of PPARα by Wy-14643 ameliorates systemic lipopolysaccharide-induced acute lung injury

Energy Technology Data Exchange (ETDEWEB)

Yoo, Seong Ho, E-mail: yoosh@snu.ac.kr [Seoul National University Hospital, Biomedical Research Institute and Institute of Forensic Medicine, Seoul National University College of Medicine, Seoul (Korea, Republic of); Abdelmegeed, Mohamed A. [Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD (United States); Song, Byoung-Joon, E-mail: bj.song@nih.gov [Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD (United States)

2013-07-05

Highlights: •Activation of PPARα attenuated LPS-mediated acute lung injury. •Pretreatment with Wy-14643 decreased the levels of IFN-γ and IL-6 in ALI. •Nitrosative stress and lipid peroxidation were downregulated by PPARα activation. •PPARα agonists may be potential therapeutic targets for acute lung injury. -- Abstract: Acute lung injury (ALI) is a major cause of mortality and morbidity worldwide. The activation of peroxisome proliferator-activated receptor-α (PPARα) by its ligands, which include Wy-14643, has been implicated as a potential anti-inflammatory therapy. To address the beneficial efficacy of Wy-14643 for ALI along with systemic inflammation, the in vivo role of PPARα activation was investigated in a mouse model of lipopolysaccharide (LPS)-induced ALI. Using age-matched Ppara-null and wild-type mice, we demonstrate that the activation of PPARα by Wy-14643 attenuated LPS-mediated ALI. This was evidenced histologically by the significant alleviation of inflammatory manifestations and apoptosis observed in the lung tissues of wild-type mice, but not in the corresponding Ppara-null mice. This protective effect probably resulted from the inhibition of LPS-induced increases in pro-inflammatory cytokines and nitroxidative stress levels. These results suggest that the pharmacological activation of PPARα might have a therapeutic effect on LPS-induced ALI.

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

International Nuclear Information System (INIS)

Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

2010-01-01

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca 2+ -dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

Uric Acid Induces Renal Inflammation via Activating Tubular NF-κB Signaling Pathway

Science.gov (United States)

Zhou, Yang; Fang, Li; Jiang, Lei; Wen, Ping; Cao, Hongdi; He, Weichun; Dai, Chunsun; Yang, Junwei

2012-01-01

Inflammation is a pathologic feature of hyperuricemia in clinical settings. However, the underlying mechanism remains unknown. Here, infiltration of T cells and macrophages were significantly increased in hyperuricemia mice kidneys. This infiltration of inflammatory cells was accompanied by an up-regulation of TNF-α, MCP-1 and RANTES expression. Further, infiltration was largely located in tubular interstitial spaces, suggesting a role for tubular cells in hyperuricemia-induced inflammation. In cultured tubular epithelial cells (NRK-52E), uric acid, probably transported via urate transporter, induced TNF-α, MCP-1 and RANTES mRNA as well as RANTES protein expression. Culture media of NRK-52E cells incubated with uric acid showed a chemo-attractive ability to recruit macrophage. Moreover uric acid activated NF-κB signaling. The uric acid-induced up-regulation of RANTES was blocked by SN 50, a specific NF-κB inhibitor. Activation of NF-κB signaling was also observed in tubule of hyperuricemia mice. These results suggest that uric acid induces renal inflammation via activation of NF-κB signaling. PMID:22761883

Cyclosporine Induces Endothelial Cell Release of Complement-Activating Microparticles

Science.gov (United States)

Renner, Brandon; Klawitter, Jelena; Goldberg, Ryan; McCullough, James W.; Ferreira, Viviana P.; Cooper, James E.; Christians, Uwe

2013-01-01

Defective control of the alternative pathway of complement is an important risk factor for several renal diseases, including atypical hemolytic uremic syndrome. Infections, drugs, pregnancy, and hemodynamic insults can trigger episodes of atypical hemolytic uremic syndrome in susceptible patients. Although the mechanisms linking these clinical events with disease flares are unknown, recent work has revealed that each of these clinical conditions causes cells to release microparticles. We hypothesized that microparticles released from injured endothelial cells promote intrarenal complement activation. Calcineurin inhibitors cause vascular and renal injury and can trigger hemolytic uremic syndrome. Here, we show that endothelial cells exposed to cyclosporine in vitro and in vivo release microparticles that activate the alternative pathway of complement. Cyclosporine-induced microparticles caused injury to bystander endothelial cells and are associated with complement-mediated injury of the kidneys and vasculature in cyclosporine-treated mice. Cyclosporine-induced microparticles did not bind factor H, an alternative pathway regulatory protein present in plasma, explaining their complement-activating phenotype. Finally, we found that in renal transplant patients, the number of endothelial microparticles in plasma increases 2 weeks after starting tacrolimus, and treatment with tacrolimus associated with increased C3 deposition on endothelial microparticles in the plasma of some patients. These results suggest that injury-associated release of endothelial microparticles is an important mechanism by which systemic insults trigger intravascular complement activation and complement-dependent renal diseases. PMID:24092930

Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action

International Nuclear Information System (INIS)

Chilampalli, Chandeshwari; Guillermo, Ruth; Zhang, Xiaoying; Kaushik, Radhey S; Young, Alan; Zeman, David; Hildreth, Michael B; Fahmy, Hesham; Dwivedi, Chandradhar

2011-01-01

Magnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development. UVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle. Magnolol pretreated groups (30, 60 μ g) before UVB treatments (30 mJ/cm 2 , 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice. Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr 705 ), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells. Magnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various

Lipocalin-2 induces NLRP3 inflammasome activation via HMGB1 induced TLR4 signaling in heart tissue of mice under pressure overload challenge.

Science.gov (United States)

Song, Erfei; Jahng, James Ws; Chong, Lisa P; Sung, Hye K; Han, Meng; Luo, Cuiting; Wu, Donghai; Boo, Stellar; Hinz, Boris; Cooper, Matthew A; Robertson, Avril Ab; Berger, Thorsten; Mak, Tak W; George, Isaac; Schulze, P Christian; Wang, Yu; Xu, Aimin; Sweeney, Gary

2017-01-01

Lipocalin-2 (also known as NGAL) levels are elevated in obesity and diabetes yet relatively little is known regarding effects on the heart. We induced pressure overload (PO) in mice and found that lipocalin-2 knockout (LKO) mice exhibited less PO-induced autophagy and NLRP3 inflammasome activation than Wt. PO-induced mitochondrial damage was reduced and autophagic flux greater in LKO mice, which correlated with less cardiac dysfunction. All of these observations were negated upon adenoviral-mediated restoration of normal lipocalin-2 levels in LKO. Studies in primary cardiac fibroblasts indicated that lipocalin-2 enhanced priming and activation of NLRP3-inflammasome, detected by increased IL-1β, IL-18 and Caspase-1 activation. This was attenuated in cells isolated from NLRP3-deficient mice or upon pharmacological inhibition of NLRP3. Furthermore, lipocalin-2 induced release of HMGB1 from cells and NLRP3-inflammasome activation was attenuated by TLR4 inhibition. We also found evidence of increased inflammasome activation and reduced autophagy in cardiac biopsy samples from heart failure patients. Overall, this study provides new mechanistic insight on the detrimental role of lipocalin-2 in the development of cardiac dysfunction.

Metformin induces differentiation in acute promyelocytic leukemia by activating the MEK/ERK signaling pathway

International Nuclear Information System (INIS)

Huai, Lei; Wang, Cuicui; Zhang, Cuiping; Li, Qihui; Chen, Yirui; Jia, Yujiao; Li, Yan; Xing, Haiyan; Tian, Zheng; Rao, Qing; Wang, Min; Wang, Jianxiang

2012-01-01

Highlights: ► Metformin induces differentiation in NB4 and primary APL cells. ► Metformin induces activation of the MEK/ERK signaling pathway in APL cells. ► Metformin synergizes with ATRA to trigger maturation of NB4 and primary APL cells. ► Metformin induces the relocalization and degradation of the PML-RARα fusion protein. ► The study may be applicable for new differentiation therapy in cancer treatment. -- Abstract: Recent studies have shown that metformin, a widely used antidiabetic agent, may reduce the risk of cancer development. In this study, we investigated the antitumoral effect of metformin on both acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL) cells. Metformin induced apoptosis with partial differentiation in an APL cell line, NB4, but only displayed a proapoptotic effect on several non-M3 AML cell lines. Further analysis revealed that a strong synergistic effect existed between metformin and all-trans retinoic acid (ATRA) during APL cell maturation and that metformin induced the hyperphosphorylation of extracellular signal-regulated kinase (ERK) in APL cells. U0126, a specific MEK/ERK activation inhibitor, abrogated metformin-induced differentiation. Finally, we found that metformin induced the degradation of the oncoproteins PML-RARα and c-Myc and activated caspase-3. In conclusion, these results suggest that metformin treatment may contribute to the enhancement of ATRA-induced differentiation in APL, which may deepen the understanding of APL maturation and thus provide insight for new therapy strategies.

Human retinal pigment epithelial cell-induced apoptosis in activated T cells

DEFF Research Database (Denmark)

Jørgensen, A; Wiencke, A K; la Cour, M

1998-01-01

PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

YB-1 overexpression promotes a TGF-β1-induced epithelial–mesenchymal transition via Akt activation

International Nuclear Information System (INIS)

Ha, Bin; Lee, Eun Byul; Cui, Jun; Kim, Yosup; Jang, Ho Hee

2015-01-01

The Y-box binding protein-1 (YB-1) is a transcription/translation regulatory protein, and the expression thereof is associated with cancer aggressiveness. In the present study, we explored the regulatory effects of YB-1 during the transforming growth factor-β1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) in lung adenocarcinoma cells. Downregulation of YB-1 increased E-cadherin promoter activity, and upregulation of YB-1 decreased promoter activity, suggesting that the YB-1 level may be correlated with the EMT. TGF-β1 induced YB-1 expression, and TGF-β1 translocated cytosolic YB-1 into the nucleus. YB-1 overexpression promoted TGF-β1-induced downregulation of epithelial markers, upregulation of mesenchymal markers, and cell migration. Moreover, YB-1 overexpression enhanced the expression of E-cadherin transcriptional repressors via TGF-β1-induced Akt activation. Our findings afford new insights into the role played by YB-1 in the TGF-β1 signaling pathway. - Highlights: • YB-1 regulates E-cadherin expression in A549 cells. • TGF-β1 induces upregulating and nuclear localization of YB-1. • YB-1 overexpression accelerates TGF-β1-induced EMT and cell migration. • YB-1 regulates Snail and Slug expression via Akt activation

δ-Tocopherol inhibits receptor tyrosine kinase-induced AKT activation in prostate cancer cells.

Science.gov (United States)

Wang, Hong; Hong, Jungil; Yang, Chung S

2016-11-01

The cancer preventive activity of vitamin E is suggested by epidemiological studies and supported by animal studies with vitamin E forms, γ-tocopherol and δ-tocopherol (δ-T). Several recent large-scale cancer prevention trials with high dose of α-tocopherol, however, yielded disappointing results. Whether vitamin E prevents or promotes cancer is a serious concern. A better understanding of the molecular mechanisms of action of the different forms of tocopherols would enhance our understanding of this topic. In this study, we demonstrated that δ-T was the most effective tocopherol form in inhibiting prostate cancer cell growth, by inducing cell cycle arrest and apoptosis. By profiling the effects of δ-T on the cell signaling using the phospho-kinase array, we found that the most inhibited target was the phosphorylation of AKT on T308. Further study on the activation of AKT by EGFR and IGFR revealed that δ-T attenuated the EGF/IGF-induced activation of AKT (via the phosphorylation of AKT on T308 induced by the activation of PIK3). Expression of dominant active PIK3 and AKT in prostate cancer cell line DU145 in which PIK3, AKT, and PTEN are wild type caused the cells to be reflectory to the inhibition of δ-T, supporting that δ-T inhibits the PIK3-mediated activation of AKT. Our data also suggest that δ-T interferes with the EGF-induced EGFR internalization, which leads to the inhibition of the receptor tyrosine kinase-dependent activation of AKT. In summary, our results revealed a novel mechanism of δ-T in inhibiting prostate cancer cell growth, supporting the cancer preventive activity δ-T. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.