Zinc Finger Nuclease Knock-out of NADPH:Cytochrome P450 Oxidoreductase (POR) in Human Tumor Cell Lines Demonstrates That Hypoxia-activated Prodrugs Differ in POR Dependence

Su, Jiechuang; Gu, Yongchuan; Pruijn, Frederik B.; Smaill, Jeff B.; Patterson, Adam V.; Guise, Christopher P.; Wilson, William R.

doi:10.1074/jbc.m113.505222

Cited by 21 publications

(27 citation statements)

References 65 publications

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“…The hypoxic cytotoxicity ratio (HCR) was calculated as the ratio of IC 50 values under aerobic conditions and the IC 50 values under hypoxic (0.1 % oxygen) conditions to illustrate selective toxicity towards cells under hypoxic conditions. Tirapazamine was used as a positive control and an HCR of 9.6 was obtained (Table ), which is in agreement with that reported in the literature . By contrast, there was no measured increase in cytotoxicity under hypoxic conditions for most test compounds, including the majority of endoperoxides ( 35 , 37 , 43 , 44 and 47 ).…”

Section: Resultssupporting

confidence: 87%

“…Tirapazamine was used as a positive control and an HCR of 9.6 was obtained (Table 1), which is in agreement with that reported in the literature. [19] By contrast, there was no measured increase in cytotoxicity under hypoxic conditions for most test compounds, including the majority of endoperoxides (35, 37, 43, 44 and 47). However, endoperoxide 40 demonstrated a statistically significantly greater activity under hypoxic compared to aerobic conditions with an HCR of 2.6.…”

Section: Pharmacology: Chemosensitivity Studiesmentioning

confidence: 92%

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Revisiting Bromohexitols as a Novel Class of Microenvironment‐Activated Prodrugs for Cancer Therapy

et al. 2019

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Bromohexitols represent a potent class of DNA‐alkylating carbohydrate chemotherapeutics that has been largely ignored over the last decades due to safety concerns. The limited structure−activity relationship data available reveals significant changes in cytotoxicity with even subtle changes in stereochemistry. However, no attempts have been made to improve the therapeutic window by rational drug design or by using a prodrug approach to exploit differences between tumour physiology and healthy tissue, such as acidic extracellular pH and hypoxia. Herein, we report the photochemical synthesis of highly substituted endoperoxides as key precursors for dibromohexitol derivatives and investigate their use as microenvironment‐activated prodrugs for targeting cancer cells. One endoperoxide was identified to have a marked increased activity under hypoxic and low pH conditions, indicating that endoperoxides may serve as microenvironment‐activated prodrugs.

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Section: Resultssupporting

confidence: 87%

Section: Pharmacology: Chemosensitivity Studiesmentioning

confidence: 92%

Revisiting Bromohexitols as a Novel Class of Microenvironment‐Activated Prodrugs for Cancer Therapy

et al. 2019

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“…1E; TCRN0000046524 > TCRN0000046526 > TCRN0000046527; henceforth shRNA 24, 26, 27 for brevity). To confirm that enrichment of hairpins against POR was an on-target effect, we screened an HCT116 clone (HKO2, HCT116-POR À/À ) in which both POR alleles were knocked out by frameshifting indels using custom-designed zinc finger nucleases (30). As expected, representation of POR shRNAs was unaffected by SN30000 treatment in HKO2 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…) POR alleles were inactivated (30). HKO1 and HKO2 cells were stably transduced with lentiviruses at an empirical MOI of 0.38 and 0.25, respectively and exposed to SN30000, after which surviving fractions were 0.008 and 0.1 (Supplementary Table S5).…”

Section: Genome-wide Shrna Screensmentioning

confidence: 99%

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Identification of P450 Oxidoreductase as a Major Determinant of Sensitivity to Hypoxia-Activated Prodrugs

et al. 2015

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Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxiaactivated prodrugs (HAP) have been developed, including the phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia. Using genome-scale shRNA screens and a high-representation library enriched for oxidoreductases, we identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in three different genetic backgrounds. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. Knockdown or genetic knockout of POR reduced SN30000 reductive metabolism and clonogenic cell death and similarly reduced sensitivity to TH-302 under hypoxia. A retrospective evaluation of head and neck squamous cell carcinomas showed heterogeneous POR expression and suggested a possible relationship between human papillomavirus status and HAP sensitivity. Taken together, our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during the clinical development of SN30000, TH-302, and other hypoxia-directed agents. Cancer Res; 75(19); 4211-23. Ó2015 AACR.

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Isotopologous Organotellurium Probes Reveal Dynamic Hypoxia In Vivo with Cellular Resolution

et al. 2016

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Changes in the oxygenation state of microenvironments within solid tumors are associated with the development of aggressive cancer phenotypes. Factors that influence cellular hypoxia have been characterized; however, methods for measuring the dynamics of oxygenation at a cellular level in vivo have been elusive. We report a series of tellurium‐containing isotopologous probes for cellular hypoxia compatible with mass cytometry (MC)—technology that allows for highly parametric interrogation of single cells based on atomic mass spectrometry. Sequential labeling with the isotopologous probes (SLIP) in pancreatic tumor xenograft models revealed changes in cellular oxygenation over time which correlated with the distance from vasculature, the proliferation of cell populations, and proximity to necrosis. SLIP allows for capture of spatial and temporal dynamics in vivo using enzyme activated probes.

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Zinc Finger Nuclease Knock-out of NADPH:Cytochrome P450 Oxidoreductase (POR) in Human Tumor Cell Lines Demonstrates That Hypoxia-activated Prodrugs Differ in POR Dependence

Cited by 21 publications

References 65 publications

Revisiting Bromohexitols as a Novel Class of Microenvironment‐Activated Prodrugs for Cancer Therapy

Revisiting Bromohexitols as a Novel Class of Microenvironment‐Activated Prodrugs for Cancer Therapy

Identification of P450 Oxidoreductase as a Major Determinant of Sensitivity to Hypoxia-Activated Prodrugs

Isotopologous Organotellurium Probes Reveal Dynamic Hypoxia In Vivo with Cellular Resolution

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