Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues

Park, Sook Ryun; Kinders, Robert J.; Khin, Sonny; Hollingshead, Melinda G.; Antony, Smitha; Parchment, Ralph E.; Tomaszewski, Joseph E.; Kummar, Shivaani; Doroshow, James H.

doi:10.1016/j.ab.2014.04.025

Cited by 11 publications

(5 citation statements)

References 35 publications

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“…Thus it is critical to select appropriate cell‐based luciferase reporter assay to screen HIF‐1 inhibitors. Notably, there is other method for the detection of the amount of HIF‐1α such as a two‐site chemiluminescent quantitative ELISA which could collect HIF‐1α in solid tumor tissues (Park et al, ). Compared to these methods, luciferase reporter assay is fast, easy, repeatable and environment‐friendly.…”

Section: Discussionmentioning

confidence: 99%

LB‐1 Exerts Antitumor Activity in Pancreatic Cancer by Inhibiting HIF‐1α and Stat3 Signaling

Niu

Lai

et al. 2015

Journal Cellular Physiology

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Hypoxia is widely present in pancreatic cancer and subsequently causes the overexpression of hypoxia-inducible factor-1α (HIF-1α) and signal transducer and activator of transcription-3 (Stat3). HIF-1α and Stat3 function cooperatively to regulate a number of downstream genes that are implicated in tumorigenesis. Thus, inhibition of HIF-1α and Stat3 is a potential therapeutic strategy for pancreatic cancer. In this study, we explored how LB-1, a novel triptolide (LA) derivative, exerted its antitumor effect through blockade of HIF-1α and Stat3 signaling. Our data showed that LB-1 was able to inhibit the proliferation and colony formation of Mia-PaCa2 and SW1990 cells. LB-1 suppressed HIF-1α protein accumulation by promoting its proteasome degradation and reducing transactivation. Moreover, the silence of HIF-1α by shRNA partially prevented the proliferation inhibition triggered by LB-1. As expected, LB-1 also decreased Stat3 protein accumulation and blocked the physical interactions between HIF-1α/p300/phosphor-Stat3 (p-Stat3) at the pharmacological concentration to reduce VEGF expression, thereby hypoxia-induced angiogenesis. In the Mia-PaCa2 nude xenograft model, therapeutic treatment with LB-1 significantly inhibited tumor growth and had minimal systemic toxicity compared to the mother drug LA. Furthermore, in accordance with in vitro results, HIF-1α activation and Stat3 expression in tumors were blocked by LB-1 through mTOR-dependent pathway. Taken together, these results illustrate that, as a potent inhibitor of HIF-1α and Stat3 signaling, LB-1 exhibits antitumor effect and could be potentially used to treat pancreatic cancer.

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Section: Discussionmentioning

confidence: 99%

LB‐1 Exerts Antitumor Activity in Pancreatic Cancer by Inhibiting HIF‐1α and Stat3 Signaling

Niu

Lai

et al. 2015

Journal Cellular Physiology

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“…For example, handling time is critical if the target molecule undergoes degradation during processing (eg, molecules in hypoxia-or phosphorylation-related pathways). 7,8 Sample preparation depends on tissue use: whole-biopsy extraction assays often require specimens to be flash frozen and then homogenized, [7][8][9][10][11] whereas slide-based analyses require frozen cores to be formalin fixed and paraffin embedded for sectioning. 12 Inadequate biopsy tumor content is also a barrier to precision medicine clinical trials in which genetic sequencing establishes trial eligibility or influences treatment selection.…”

Section: Introductionmentioning

confidence: 99%

What Can Be Done to Improve Research Biopsy Quality in Oncology Clinical Trials?

Ferry-Galow

Datta

Makhlouf

et al. 2018

JOP

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Purpose: Research biopsy specimens collected in clinical trials often present requirements beyond those of tumor biopsy specimens collected for diagnostic purposes. Research biopsies underpin hypothesis-driven drug development, pharmacodynamic assessment of molecularly targeted anticancer agents, and, increasingly, genomic assessment for precision medicine; insufficient biopsy specimen quality or quantity therefore compromises the scientific value of a study and the resources devoted to it, as well as each patient’s contribution to and potential benefit from a clinical trial. Methods: To improve research biopsy specimen quality, we consulted with other translational oncology teams and reviewed current best practices. Results: Among the recommendations were improving communication between oncologists and interventional radiologists, providing feedback on specimen sufficiency, increasing academic recognition and financial support for the time investment required by radiologists to collect and preserve research biopsy specimens, and improving real-time assessment of tissue quality. Conclusion: Implementing these recommendations at the National Cancer Institute’s Developmental Therapeutics Clinic has demonstrably improved the quality of biopsy specimens collected; more widespread dissemination of these recommendations beyond large clinical cancer centers is possible and will be of value to the community in improving clinical research and, ultimately, patient care.

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“…Due to the need for a more precise method for HIF-1α quantification in biological samples, some quantitative methods have also been developed, that include the enzyme linked immunosorbent assay (ELISA) (Formento et al 2005 ). Although it has been reported that HIF-1α is detectable in plasma, (He et al 2016a ) it is unstable and is rapidly degraded under normal oxygen tensions, as discussed above (Park et al 2014 ). As a transcription factor, it is normally located within the cell, and there has also been some doubt about its release into plasma (Ferns and Heikal 2017 ).…”

Section: Introductionmentioning

confidence: 96%

“…Several semi-quantitative methods have been used to estimate HIF-1α levels, for example Western blotting and immunocytochemistry (Karshovska et al 2007 ; Srinivasan and Dunn 2011 ). HIF-1α levels have also been assessed indirectly by quantifying mRNA expression, or by measuring downstream target genes such as VEGF and EPO (Formento et al 2005 ; Park et al 2014 ).…”

Section: Introductionmentioning

confidence: 99%

Assessment of HIF-1α expression and release following endothelial injury in-vitro and in-vivo

Heikal

Ghezzi

Mengozzi

et al. 2018

Mol Med

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BackgroundEndothelial injury is an early and enduring feature of cardiovascular disease. Inflammation and hypoxia may be responsible for this, and are often associated with the up-regulation of several transcriptional factors that include Hypoxia Inducible Factor-1 (HIF-1). Although it has been reported that HIF-1α is detectable in plasma, it is known to be unstable. Our aim was to optimize an assay for HIF-1α to be applied to in vitro and in vivo applications, and to use this assay to assess the release kinetics of HIF-1α following endothelial injury.MethodsAn ELISA for the measurement of HIF-1α in cell-culture medium and plasma was optimized, and the assay was used to determine the best conditions for sample collection and storage. The results of the ELISA were validated using Western blotting and immunohistochemistry (IHC). In vitro, a standardized injury was produced in a monolayer of rat aortic endothelial cells (RAECs) and intracellular HIF-1α was measured at intervals over 24 h. In vivo, a rat angioplasty model was used. The right carotid artery was injured using a 2F Fogarty balloon catheter. HIF-1α was measured in the plasma and in the arterial tissue (0, 1, 2, 3 and 5 days post injury).ResultsThe HIF-1α ELISA had a limit of detection of 2.7 pg/mL and was linear up to 1000 pg/ mL. Between and within-assay, the coefficient of variation values were less than 15%. HIF-1α was unstable in cell lysates and plasma, and it was necessary to add a protease inhibitor immediately after collection, and to store samples at -80 °C prior to analysis. The dynamics of HIF-1α release were different for the in vitro and in vivo models. In vitro, HIF-1α reached maximum concentrations approximately 2 h post injury, whereas peak values in plasma and tissues occurred approximately 2 days post injury, in the balloon injury model.ConclusionHIF-1α can be measured in plasma, but this requires careful sample collection and storage. The carotid artery balloon injury model is associated with the transient release of HIF-1α into the circulation that probably reflects the hypoxia induced in the artery wall.Electronic supplementary materialThe online version of this article (10.1186/s10020-018-0026-5) contains supplementary material, which is available to authorized users.

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Validation of a hypoxia-inducible factor-1 alpha specimen collection procedure and quantitative enzyme-linked immunosorbent assay in solid tumor tissues

Cited by 11 publications

References 35 publications

LB‐1 Exerts Antitumor Activity in Pancreatic Cancer by Inhibiting HIF‐1α and Stat3 Signaling

LB‐1 Exerts Antitumor Activity in Pancreatic Cancer by Inhibiting HIF‐1α and Stat3 Signaling

What Can Be Done to Improve Research Biopsy Quality in Oncology Clinical Trials?

Assessment of HIF-1α expression and release following endothelial injury in-vitro and in-vivo

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