Hypoxic human cancer cells are sensitized to BH-3 mimetic–induced apoptosis via downregulation of the Bcl-2 protein Mcl-1
Luke R.E. Harrison, … , Guy Makin, Caroline Dive
Luke R.E. Harrison, … , Guy Makin, Caroline Dive
Published February 14, 2011
Citation Information: J Clin Invest. 2011;121(3):1075-1087. https://doi.org/10.1172/JCI43505.
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Research Article Oncology

Hypoxic human cancer cells are sensitized to BH-3 mimetic–induced apoptosis via downregulation of the Bcl-2 protein Mcl-1

Abstract

Solid tumors contain hypoxic regions in which cancer cells are often resistant to chemotherapy-induced apoptotic cell death. Therapeutic strategies that specifically target hypoxic cells and promote apoptosis are particularly appealing, as few normal tissues experience hypoxia. We have found that the compound ABT-737, a Bcl-2 homology domain 3 (BH-3) mimetic, promotes apoptotic cell death in human colorectal carcinoma and small cell lung cancer cell lines exposed to hypoxia. This hypoxic induction of apoptosis was mediated through downregulation of myeloid cell leukemia sequence 1 (Mcl-1), a Bcl-2 family protein that serves as a biomarker for ABT-737 resistance. Downregulation of Mcl-1 in hypoxia was independent of hypoxia-inducible factor 1 (HIF-1) activity and was consistent with decreased global protein translation. In addition, ABT-737 induced apoptosis deep within tumor spheroids, consistent with an optimal hypoxic oxygen tension being necessary to promote ABT-737–induced cell death. Tumor xenografts in ABT-737–treated mice also displayed significantly more apoptotic cells within hypoxic regions relative to normoxic regions. Synergies between ABT-737 and other cytotoxic drugs were maintained in hypoxia, suggesting that this drug may be useful in combination with chemotherapeutic agents. Taken together, these findings suggest that Mcl-1–sparing BH-3 mimetics may induce apoptosis in hypoxic tumor cells that are resistant to other chemotherapeutic agents and may have a role in combinatorial chemotherapeutic regimens for treatment of solid tumors.

Authors

Luke R.E. Harrison, Dimitra Micha, Martin Brandenburg, Kathryn L. Simpson, Christopher J. Morrow, Olive Denneny, Cassandra Hodgkinson, Zaira Yunus, Clare Dempsey, Darren Roberts, Fiona Blackhall, Guy Makin, Caroline Dive

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Figure 6

Mechanism of Mcl-1 loss in hypoxia.

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Mechanism of Mcl-1 loss in hypoxia. (A) HCT116 cells were treated with N...
(A) HCT116 cells were treated with NT or MULE siRNA before incubation in either normoxia or hypoxia for 6 hours, after which cells were harvested and samples analyzed for expression of MULE, Mcl-1, and GAPDH by Western blot. (B) HCT116 cells were incubated in hypoxia for up to 24 hours and were harvested at various time points for measurement of Mcl-1 levels by Western blot followed by densitometry analysis of Mcl-1, results of which were then plotted as a function of time. (C) HCT116 cells were incubated in hypoxia or normoxia for 4 hours, after which cycloheximide (10 μg/ml) was added and cells harvested every 20 minutes for the next 2 hours. Samples were then analyzed as in B. (D) Cells were incubated in hypoxia or normoxia for 6 hours, after which MG132 (10 μM) was added and cells were harvested at various time points and analyzed as in B. (E) qRT-PCR analysis of MCL1 mRNA after 18 hours preincubation in normoxia and hypoxia; results were normalized to housekeeping genes. (F) Lysates from hypoxic and normoxic cells (3-hour incubation) were separated by density on a 10%–60% sucrose gradient before being fractionated into non-polysomal (fractions 1–9) or polysomal (fractions 10–18) fractions and subjected to OD254 measurement. Data are mean ± SEM of 3 independent experiments.