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Reverse-phase protein array

Uday B Maachani, Uma Shankavaram, Tamalee Kramp, Philip J Tofilon, Kevin Camphausen, Anita T Tandle
Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We identified subsets of proteins with clearly concordant/discordant behavior between irradiated and non-irradiated GBM cells in vitro and in vivo...
October 14, 2016: Oncotarget
Sofia Lisanti, David S Garlick, Kelly G Bryant, Michele Tavecchio, Gordon B Mills, Yiling Lu, Andrew V Kossenkov, Louise C Showe, Lucia R Languino, Dario C Altieri
Protein homeostasis, or proteostasis is required for mitochondrial function, but its role in cancer is controversial. Here, we show that transgenic mice expressing the mitochondrial chaperone, TRAP1 in the prostate develop epithelial hyperplasia and cellular atypia. When examined on a Pten+/- background, a common alteration in human prostate cancer, TRAP1 transgenic mice showed accelerated incidence of invasive prostatic adenocarcinoma, characterized by increased cell proliferation and reduced apoptosis, in situ...
October 17, 2016: Journal of Biological Chemistry
Schammim Ray Amith, Krista Marie Vincent, Jodi Marie Wilkinson, Lynne Marie Postovit, Larry Fliegel
Mounting evidence supports a major role for the Na(+)/H(+) exchanger NHE1 in cancer progression and metastasis. NHE1 is hyperactive at the onset of oncogenic transformation, resulting in intracellular alkalinization and extracellular microenvironmental acidification. These conditions promote invasion and facilitate metastasis. However, the signal pathways governing the regulation of exchanger activity are still unclear. This is especially important in the aggressively metastatic, triple-negative basal breast cancer subtype...
October 15, 2016: Cellular Signalling
D T Saenz, W Fiskus, T Manshouri, K Rajapakshe, S Krieger, B Sun, C P Mill, C DiNardo, N Pemmaraju, T Kadia, S Parmar, S Sharma, C Coarfa, P Qiu, S Verstovsek, K N Bhalla
Myeloproliferative Neoplasms with myelofibrosis (MPN-MF) demonstrate constitutive activation of JAK-STAT signaling, which responds to treatment with the JAK1 & 2 kinase inhibitor (JAKi) ruxolitinib. However, MPN-MF often progresses (~20%) to secondary AML (sAML), where standard induction chemotherapy or ruxolitinib is relatively ineffective, necessitating the development of novel therapeutic approaches. In the present studies, we demonstrate that treatment with BET (bromodomain and extra terminal) protein inhibitor (BETi), e...
September 28, 2016: Leukemia: Official Journal of the Leukemia Society of America, Leukemia Research Fund, U.K
Yiling Lu, Shiyun Ling, Apurva M Hegde, Lauren A Byers, Kevin Coombes, Gordon B Mills, Rehan Akbani
The majority of the targeted therapeutic agents in clinical use target proteins and protein function. Although DNA and RNA analyses have been used extensively to identify novel targets and patients likely to benefit from targeted therapies, these are indirect measures of the levels and functions of most therapeutic targets. More importantly, DNA and RNA analysis is ill-suited for determining the pharmacodynamic effects of target inhibition. Assessing changes in protein levels and function is the most efficient way to evaluate the mechanisms underlying sensitivity and resistance to targeted agents...
August 2016: Seminars in Oncology
C Petit, F Gouel, I Dubus, C Heuclin, K Roget, J P Vannier
BACKGROUND: Several studies show that bone marrow (BM) microenvironment and hypoxia condition can promote the survival of leukemic cells and induce resistance to anti-leukemic drugs. However, the molecular mechanism for chemoresistance by hypoxia is not fully understood. METHODS: In the present study, we investigated the effect of hypoxia on resistance to two therapies, methotrexate (MTX) and prednisolone (PRD), in two cell models for acute lymphoblastic leukemia (ALL)...
2016: BMC Cancer
Nathan Moerke, Mohammad Fallahi-Sichani
Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post-translational modifications of interest. RPPA assays are well suited for large-scale, high-throughput measurement of protein and PTM levels in cells and tissues. RPPAs are affordable and highly multiplexable, as a large number of arrays can readily be produced in parallel and then probed separately with distinct primary antibodies...
2016: Current Protocols in Chemical Biology
Hani Lee, Yonghwan Kim, Ji Hye Jeong, Jae-Ha Ryu, Woo-Young Kim
Among the many stilbenoids found in a variety of berries, resveratrol and pterostilbene are of particular interest given their potential for use in cancer therapeutics and prevention. We purified four stilbenoids from R. undulatum and found that pterostilbene inhibits cancer cell proliferation more efficiently than rhapontigenin, piceatannol and resveratrol. To investigate the underlying mechanism of this superior action of pterostilbene on cancer cells, we utilized a reverse-phase protein array followed by bioinformatic analysis and found that the ATM/CHK pathway is modified by pterostilbene in a lung cancer cell line...
2016: PloS One
Merve Mutlu, Özge Saatci, Suhail A Ansari, Emre Yurdusev, Huma Shehwana, Özlen Konu, Umar Raza, Özgür Şahin
Dysregulation of PI3K and MAPK pathways promotes uncontrolled cell proliferation, apoptotic inhibition and metastasis. Individual targeting of these pathways using kinase inhibitors has largely been insufficient due to the existence of cross-talks between these parallel cascades. MicroRNAs are small non-coding RNAs targeting several genes simultaneously and controlling cancer-related processes. To identify miRNAs repressing both PI3K and MAPK pathways in breast cancer, we re-analyzed our previous miRNA mimic screen data with reverse phase protein array (RPPA) output, and identified miR-564 inhibiting both PI3K and MAPK pathways causing markedly decreased cell proliferation through G1 arrest...
2016: Scientific Reports
Dena A J Ahmad, Ola H Negm, M Layth Alabdullah, Sameer Mirza, Mohamed R Hamed, Vimla Band, Andrew R Green, Ian O Ellis, Emad A Rakha
BACKGROUND: Mitogen-activated protein kinases (MAPKs) are signalling transduction molecules that have different functions and diverse behaviour in cancer. In breast cancer, MAPK is related to oestrogen receptor (ER) and HER2. METHODS: Protein expression of a large panel of MAPKs (JNK1/2, ERK, p38, C-JUN and ATF2 including phosphorylated forms) were assessed immunohistochemically in a large (n = 1400) and well-characterised breast cancer series prepared as tissue microarray...
October 2016: Breast Cancer Research and Treatment
Toshihiro Yoneyama, Sumio Ohtsuki, Kazufumi Honda, Makoto Kobayashi, Motoki Iwasaki, Yasuo Uchida, Takuji Okusaka, Shoji Nakamori, Masashi Shimahara, Takaaki Ueno, Akihiko Tsuchida, Naohiro Sata, Tatsuya Ioka, Yohichi Yasunami, Tomoo Kosuge, Takashi Kaneda, Takao Kato, Kazuhiro Yagihara, Shigeyuki Fujita, Wilber Huang, Tesshi Yamada, Masanori Tachikawa, Tetsuya Terasaki
Pancreatic cancer is one of the most lethal tumors, and reliable detection of early-stage pancreatic cancer and risk diseases for pancreatic cancer is essential to improve the prognosis. As 260 genes were previously reported to be upregulated in invasive ductal adenocarcinoma of pancreas (IDACP) cells, quantification of the corresponding proteins in plasma might be useful for IDACP diagnosis. Therefore, the purpose of the present study was to identify plasma biomarkers for early detection of IDACP by using two proteomics strategies: antibody-based proteomics and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics...
2016: PloS One
Salah-Eddine Lamhamedi-Cherradi, Brian A Menegaz, Vandhana Ramamoorthy, Deeksha Vishwamitra, Ying Wang, Rebecca L Maywald, Adriana S Buford, Izabela Fokt, Stanislaw Skora, Jing Wang, Aung Naing, Alexander J Lazar, Eric M Rohren, Najat C Daw, Vivek Subbiah, Robert S Benjamin, Ravin Ratan, Waldemar Priebe, Antonios G Mikos, Hesham M Amin, Joseph A Ludwig
BACKGROUND: Therapies cotargeting insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) have demonstrated remarkable, albeit short-lived, clinical responses in a subset of Ewing sarcoma (ES) patients. However, the mechanisms of resistance and applicable strategies for overcoming drug resistance to the IGF-1R/mTOR blockade are still undefined. METHODS: To elucidate predominant mechanism(s) of acquired drug resistance while identifying synergistic drug combinations that improve clinical efficacy, we generated more than 18 ES cell lines resistant to IGF-1R- or mTOR-targeted therapy...
December 2016: Journal of the National Cancer Institute
Peiyan Liu, Yueling Sun, Guangbin Qiu, Hongkun Jiang, Guangrong Qiu
Congenital heart diseases (CHDs) are the most common birth defects due to abnormal cardiac development. The T-box 20 (TBX20) gene is a member of the T‑box family of transcription factors and encodes TBX20, which is essential for early heart development. In the present study, reduced TBX20 expression was observed in CHD tissue samples compared with normal tissues, and the function of TBX20 in Rattus norvegicus myocardial cells [H9c2(2-1)] and human embryonic kidney cells (HEK293) was investigated. TBX20 was silenced in H9c2 and HEK293 cells via transfection of small interfering RNA and short hairpin RNA duplexes, respectively, and TBX20 mRNA and protein levels were subsequently examined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis...
October 2016: Molecular Medicine Reports
Julian Biau, Emmanuel Chautard, Frank Court, Bruno Pereira, Pierre Verrelle, Flavien Devun, Leanne De Koning, Marie Dutreix
Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology...
August 2016: Translational Oncology
Cuicui Lv, Ganye Zhao, Xinpei Sun, Pan Wang, Nan Xie, Jianyuan Luo, Tanjun Tong
Forkhead box transcription factor M1 (FOXM1) plays crucial roles in a wide array of biological processes, including cell proliferation and differentiation, the cell cycle, and tumorigenesis by regulating the expression of its target genes. Elevated expression of FOXM1 is frequently observed in a multitude of malignancies. Here we show that FOXM1 can be acetylated by p300/CBP at lysines K63, K422, K440, K603 and K614 in vivo. This modification is essential for its transactivation on the target genes. Acetylation of FOXM1 increases during the S phase and remains high throughout the G2 and M phases, when FOXM1 transcriptional activity is required...
August 17, 2016: Oncotarget
Sara Serra, Silvia Deaglio
This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5'-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention...
2016: Journal of Visualized Experiments: JoVE
Guillaume Brachet, Thomas Bourquard, Nathalie Gallay, Eric Reiter, Valérie Gouilleux-Gruart, Anne Poupon, Hervé Watier
Eculizumab is an anti-complement C5 monoclonal antibody which has greatly improved the prognosis and outcomes of nocturnal paroxysmal hemoglobinuria and atypical hemolytic and uremic syndromes. It is also known to be very species-specific for human C5, despite an important degree of conservation of the targeted macroglobulin domain, MG7, with that of other primates. However, the published eculizumab linear epitope does not explain this species specificity. Sequence analysis, in silico docking and reverse phase protein array were implemented to fully characterize the eculizumab epitope on human complement C5...
September 2016: Molecular Immunology
Colin T Mant, Robert S Hodges
Reversed-phase high-performance liquid chromatography (RP-HPLC) is of fundamental importance to the isolation and separation of peptides, proteins, and other biomolecules. Hence, there is a continuing high demand for the development of RP-HPLC stationary-phase materials with enhanced separation efficiency. HALO packing materials began the revolution in "core-shell" technology with the advantages of faster separations, higher resolution and peak capacity, high temperature stability, and rugged reliable performance compared to traditional HPLC and UHPLC...
2016: Current Protocols in Protein Science
Alaa Tarig Alshareeda, Ola H Negm, Mohammed A Aleskandarany, Andrew R Green, Christopher Nolan, Patrick J TigHhe, Srinivasan Madhusudan, Ian O Ellis, Emad A Rakha
Impaired DNA damage response (DDR) may play a fundamental role in the pathogenesis of breast cancer (BC). RAD51 is a key player in DNA double-strand break repair. In this study, we aimed to assess the biological and clinical significance of RAD51 expression with relevance to different molecular classes of BC and patients' outcome. The expression of RAD51 was assessed immunohistochemically in a well-characterised annotated series (n = 1184) of early-stage invasive BC with long-term follow-up. A subset of cases of BC from patients with known BRCA1 germline mutations was included as a control group...
August 2016: Breast Cancer Research and Treatment
Zhihong Zeng, Rui-Yu Wang, Yi Hua Qiu, Duncan H Mak, Kevin Coombes, Suk Young Yoo, Qi Zhang, Katti Jessen, Yi Liu, Christian Rommel, David A Fruman, Hagop M Kantarjian, Steven M Kornblau, Michael Andreeff, Marina Konopleva
mTOR activation leads to enhanced survival signaling in acute myeloid leukemia (AML) cells. The active-site mTOR inhibitors (asTORi) represent a promising new approach to targeting mTOR in AKT/mTOR signaling. MLN0128 is an orally-administered, second-generation asTORi, currently in clinical development. We examined the anti-leukemic effects and the mechanisms of action of MLN0128 in AML cell lines and primary samples, with a particular focus on its effect in AML stem/progenitor cells. MLN0128 inhibited cell proliferation and induced apoptosis in AML by attenuating the activity of mTOR complex 1 and 2...
July 4, 2016: Oncotarget
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