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. 2008 Apr 15;68(8):2813-9.
doi: 10.1158/0008-5472.CAN-08-0053.

The mitochondrial uncoupling protein-2 promotes chemoresistance in cancer cells

Zoltan Derdak  1 , Nicholas M Mark, Guido Beldi, Simon C Robson, Jack R Wands, György Baffy
Affiliations

The mitochondrial uncoupling protein-2 promotes chemoresistance in cancer cells

Zoltan Derdak et al. Cancer Res. .

Abstract

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. This survival process is facilitated through efficient control of oxidative stress originating from mitochondria that typically initiates programmed cell death. We show this critical adaptive response in cancer cells to be linked to uncoupling protein-2 (UCP2), a mitochondrial suppressor of reactive oxygen species (ROS). UCP2 is present in drug-resistant lines of various cancer cells and in human colon cancer. Overexpression of UCP2 in HCT116 human colon cancer cells inhibits ROS accumulation and apoptosis after exposure to chemotherapeutic agents. Tumor xenografts of UCP2-overexpressing HCT116 cells retain growth in nude mice receiving chemotherapy. Augmented cancer cell survival is accompanied by altered NH(2)-terminal phosphorylation of the pivotal tumor suppressor p53 and induction of the glycolytic phenotype (Warburg effect). These findings link UCP2 with molecular mechanisms of chemoresistance. Targeting UCP2 may be considered a novel treatment strategy for cancer.

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Figures

Figure 1
Figure 1. Overexpression of UCP2 in cancer cells
(A) Cell line selection for overexpression experiments. Endogenous UCP2 mRNA levels in various human colon cancer cell lines determined by quantitative real time PCR and expressed as relative ratios over the mRNA of TATA-box binding protein shown in arbitrary units ± SEM. (B) Immunoblot analysis of UCP2 in the mitochondrial fraction of HCT116 cells transfected with various amounts of hUCP2-pcDNA3.1/Zeo(−) plasmid containing the full-length human ucp2 cDNA (pUCP2) or with empty vector (EV) indicates dose-dependent expression of plasmid-encoded UCP2, while endogenous UCP2 protein in these cells is essentially non-detectable. Subunit IV of cytochrome c oxidase (COX IV) served as loading control. (C) Plasmid-encoded UCP2 is properly targeted to the mitochondrial inner membrane (IM). Mitochondria of HCT116 cells were isolated and sub-fractionated 48 hours after transfection with 2 μg of plasmid. Immunoblotting indicates the presence of plasmid-encoded UCP2 in IM fraction (identified by COX IV), but not in the inter-membrane space (identified by cytochrome c). OM, mitochondrial outer membrane. (D) Functional analysis of plasmid-encoded UCP2. Left, mitochondrial membrane potential (Δψm) of HCT116 cells in response to UCP2 overexpression assessed by red to green JC-1 fluorescence ratios and shown in percentages (± SEM) relative to non-transfected cells (no DNA). Experimental controls to abolish or elevate Δψm included the chemical uncoupler FCCP (10 μM) and the ATP synthase inhibitor oligomycin (10 μM), respectively. Middle, oxygen consumption (pmol min−1 cell−1 ± SEM) measured by polarography using a Clark-type oxygen-sensitive electrode. Right, intracellular ATP content (pmol 103 cells−1 ± SEM) measured by luciferin-luciferase assay. *P < 0.05 for differences between pUCP2 vs. EV.
Figure 2
Figure 2. UCP2 inhibits apoptosis and decreases ROS levels in cancer cells
HCT116 human colon cancer cells without transfection (no DNA), transfected with 2 μg empty vector (EV), or 2 μg hUCP2-pcDNA3.1/Zeo(−) plasmid (pUCP2) were exposed to camptothecin (CPT, 2.5 μM), etoposide (10 μM), or doxorubicin (doxo, 20 μM) for 24 hours except as indicated. (A) Analysis of CPT-induced apoptosis. Left, cells were stained with FITC-conjugated annexin V and propidium iodide (PI). Results are shown in dot plots with 4-decade log scale with percentages of apoptotic cells (lower right quadrant). Right top, DNA fragmentation was assessed by accelerated DNA ladder gel electrophoresis. Marker, DNA molecular weight control. Right bottom, immunoblot analysis of pro-apoptotic (full and cleaved caspase-3, PUMA-α) and anti-apoptotic proteins (Bcl-XL). (B) Impact of UCP2 overexpression on cellular responses to various cytotoxic drugs. Top, mean rates of apoptosis (± SEM) are expressed as the percentage of total cell number assessed by annexin V flow cytometry. Bottom, intracellular ROS levels assessed by DCF flow cytometry. DCF fluorescence is expressed as the percentage of levels measured in non-transfected, untreated cells (mean ± SEM) at baseline and following treatment for 30 min. *P < 0.05 for difference between pUCP2 vs. EV. All treatments were initiated 24 hours after transfection.
Figure 3
Figure 3. Uncoupling mimics the effect of UCP2 in cancer cells
(A) At the doses indicated, HCT116 cells were treated with the protonophore carbonylcyanide-4-trifluoro-methoxyphenylhydrazone (FCCP) 30 min prior to the addition of CPT for 24 hours. Apoptosis was assessed by (left) annexin V staining, (right top) DNA ladder formation, (right middle) caspase-3 cleavage, and (right bottom) disappearance of Bcl-XL. For additional details, please see Figure 1. Note increased number of cells staining for both annexin V and propidium iodide (PI) in response to 5 μM FCCP (and at higher doses, not shown) in the right upper quadrants, indicating concomitant increase in necrotic cell death. Results are each from at least two independent experiments. (B) Intracellular ROS levels (mean ± SEM, expressed as the percentage of levels measured by DCF in untreated cells) at baseline and in response to treatment with 2.5 μM CPT for 30 min. ROS levels in cells treated with the antioxidant N-acetylcysteine (NAC, 2.5 mM) are shown for comparison. (C) Intracellular ATP content (pmol 103 cells−1 ± SEM) measured by luciferin-luciferase assay shows dose-dependent decrease following treatment with FCCP, but FCCP has no further effect on markedly decreased ATP levels in cells exposed to 2.5 μg camptothecin (CPT) for 24 hours. *P < 0.05 for differences between cells with or without treatment with CPT; ‡ P < 0.05 for differences between cells with or without treatment with FCCP.
Figure 4
Figure 4. UCP2 promotes in vivo drug resistance in cancer cells
(A) Detection of UCP2 expression by immunoblot analysis (top) and immunohistochemistry (bottom) in subcutaneous xenografts of HCT116 cells stably expressing UCP2 (ZU7, right panel) or empty vector controls (ZE12, left panel) inoculated at a dose of 3 × 106 cells into both flanks of 4-6 weeks old male NCr nu/nu mice (n = 8-8). ZE12 cells with scattered and faintly positive staining reflect endogenous UCP2 expression. β-actin served as loading control. Magnification, 400X. (B) Growth of HCT116 cancer cell xenografts monitored by triaxial measurements. Two weeks after inoculation of HCT116 cells, mice were treated with irinotecan (CPT-11) at a dose of 25 mg/kg i.p. every 3 days (arrows). Controls received saline injection. *P < 0.05 for difference between ZU7 vs. ZE12 following CPT-11 treatment.
Figure 5
Figure 5. UCP2 interferes with p53 responses in cancer cells
(A) Cell cycle analysis of p53+/+ and p53−/− HCT116 cells with no transfection (no DNA), transfected with 2 μg empty vector (EV), or with 2 μg hUCP2-pcDNA3.1/Zeo (−) plasmid (pUCP2) and treated with CPT (2.5 μM) for 24 hours. Sub-G1 fraction is indicated as apoptosis. (B) Immunoblot analysis of post-translational modification and accumulation of p53 in HCT116 cells overexpressing UCP2 (pUCP2) treated with CPT (2.5 μM) for 24 hours. N-terminal phosphorylation of p53 at selected serine residues (Ser15, Ser33, Ser46) responsive to oxidative stress and abundance of total p53 is shown. β-actin served as loading control. (C) Glycolytic activity was assessed by measuring lactate levels in the medium of HCT116 cells stably expressing UCP2 (ZU7) or empty vector controls (ZE12) at various times after seeding into culture. (D) Suppression of cell growth by inhibiting glycolysis in HCT116 cells plated 24h prior to the addition of 50 mM 2-deoxyglucose. Numbers of viable cells are expressed in percentage of pretreatment counts. *P < 0.01; ‡P < 0.0001 for differences between ZU7 vs. ZE12.
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