Biochemical Journal

Research article

Increased levels of superoxide and H2O2 mediate the differential susceptibility of cancer cells versus normal cells to glucose deprivation

Nùkhet Aykin-Burns, Iman M. Ahmad, Yueming Zhu, Larry W. Oberley, Douglas R. Spitz

Abstract

Cancer cells, relative to normal cells, demonstrate increased sensitivity to glucose-deprivation-induced cytotoxicity. To determine whether oxidative stress mediated by O2•− and hydroperoxides contributed to the differential susceptibility of human epithelial cancer cells to glucose deprivation, the oxidation of DHE (dihydroethidine; for O2•−) and CDCFH2 [5- (and 6-)carboxy-2′,7′-dichlorodihydrofluorescein diacetate; for hydroperoxides] was measured in human colon and breast cancer cells (HT29, HCT116, SW480 and MB231) and compared with that in normal human cells [FHC cells, 33Co cells and HMECs (human mammary epithelial cells)]. Cancer cells showed significant increases in DHE (2–20-fold) and CDCFH2 (1.8–10-fold) oxidation, relative to normal cells, that were more pronounced in the presence of the mitochondrial electron-transport-chain blocker, antimycin A. Furthermore, HCT116 and MB231 cells were more susceptible to glucose-deprivation-induced cytotoxicity and oxidative stress, relative to 33Co cells and HMECs. HT29 cells were also more susceptible to 2DG (2-deoxyglucose)-induced cytotoxicity, relative to FHC cells. Overexpression of manganese SOD (superoxide dismutase) and mitochondrially targeted catalase significantly protected HCT116 and MB231 cells from glucose-deprivation-induced cytotoxicity and oxidative stress and also protected HT29 cells from 2DG-induced cytotoxicity. These results show that cancer cells (relative to normal cells) demonstrate increased steady-state levels of ROS (reactive oxygen species; i.e. O2•− and H2O2) that contribute to differential susceptibility to glucose-deprivation-induced cytotoxicity and oxidative stress. These studies support the hypotheses that cancer cells increase glucose metabolism to compensate for excess metabolic production of ROS and that inhibition of glucose and hydroperoxide metabolism may provide a biochemical target for selectively enhancing cytotoxicity and oxidative stress in human cancer cells.

  • cancer cell
  • fluorescent dye
  • glucose deprivation
  • H2O2
  • metabolic oxidative stress
  • superoxide dismutase

INTRODUCTION

For more than eight decades, it has been known that cancer cells exhibit altered metabolism when compared with their normal counterparts [14]. These alterations include increased rates of glycolysis and pentose phosphate cycle activity along with slightly reduced mitochondrial respiration [14]. Originally, these metabolic abnormalities were thought to result from some impairment or damage to the cancer cell's ability to undergo respiration [2], but the underlying mechanisms responsible for these metabolic abnormalities are still not well understood.

In normal mammalian cells, mitochondria represent the major cellular organelle responsible for respiration [1]. ETCs (electron transport chains) in the mitochondrial inner membrane are believed to be responsible for most of the cellular O2 consumption and are hypothesized to be a source of ROS (reactive oxygen species) during metabolism [59]. In normal cells, as much as 1% of the electrons flowing through ETCs are thought to undergo one-electron reductions of O2 to form superoxide (O2•−), which can then react to form other ROS such as H2O2 and organic hydroperoxides [5,1013]. However, there is a lack of studies directly comparing steady-state levels of O2•− in cancer versus normal epithelial cells.

Studies of cancer cell mitochondria have noted many structural abnormalities [14,15], and epithelial cancers from colon and breast have also demonstrated higher rates of mutations in mitochondrial DNA [16]. These results have led to the speculation that cancer cells may have mitochondrial ETC defects that could contribute to increased steady-state levels of O2•− and H2O2 in human tumour cells when compared with normal cells, but direct evidence supporting this speculation is lacking [1013,16,17]. In addition, previous studies using glucose deprivation have suggested that increases in glucose metabolism in cancer cells, relative to normal cells, could be necessary to provide reducing equivalents in the form of NADPH and pyruvate for the detoxification of ROS [1013]. However, there is no direct evidence linking increased steady-state levels of O2•− and H2O2 to the differential susceptibility of epithelial cancer versus normal cells to glucose-deprivation-induced cytotoxicity and oxidative stress.

In the present study, carcinoma cells from human colon and breast epithelial tissue are shown to have increased steady-state levels of endogenous O2•− and ROS compared with normal cells derived from the same tissues, as well as consuming more glucose, having higher activities of pentose phosphate pathway enzymes associated with NADPH regeneration, and being more sensitive to oxidative stress and cell killing induced by glucose deprivation or treatment with an inhibitor of glucose metabolism [2DG (2-deoxyglucose)]. Furthermore, glucose-deprivation- or 2DG-induced cytotoxicity as well as parameters indicative of oxidative stress could be inhibited by co-overexpression of mitochondrially targeted SOD (superoxide dismutase) [MnSOD (Mn-containing SOD)] and mitoCAT (mitochondrially targeted catalase), which scavenge O2•− and H2O2 respectively. The results demonstrate that metabolic oxidative stress mediated by O2•− and H2O2 significantly contributes to the differential susceptibility of cancer versus normal epithelial cells to glucose-deprivation- or 2DG-induced cytotoxicity. These results support the hypothesis that cancer cells exhibit increased glucose metabolism to compensate for excess metabolic production of ROS, as well as the hypothesis that inhibition of glucose and hydroperoxide metabolism may provide a biochemical target for selectively enhancing cytotoxicity and oxidative stress in human cancer cells.

MATERIALS AND METHODS

Cell and culture conditions

HT29, HCT116 and SW480 human colon carcinoma cells were obtained from the A.T.C.C. (Manassas, VA, U.S.A.); MDA-MB231 breast cancer cells were a gift from Dr Mary Hendrix (Northwestern University). Cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS (fetal bovine serum; HyClone, Logan, UT, U.S.A.). Normal non-immortalized HMECs (human mammary epithelial cells) were purchased from Clonetics (East Rutherford, NJ, U.S.A.) and the cells were maintained in MEBM (mammary epithelium basal medium; Clonetics). Normal non-immortalized human colon cells, namely FHC (epithelial) and CCD-33Co (fibroblasts), were obtained from the A.T.C.C. 33Co cells were maintained in EMEM (Eagle's minimal essential medium) with 2 mM L-glutamine and Earle's balanced salt solution with 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate and 10% FBS. FHC cells were grown in Dulbecco's modified Eagle's medium and F-12 medium (1:1; v/v) supplemented with 10% FBS, insulin (5 mg/ml), cortisol (0.5 mg/ml), transferrin (50 mg/ml), cholera toxin (500 ng/μl) and Hepes (20 mM). Normal (non-immortalized) human cells (passages 2–5) were maintained in a humidified 4% O2 chamber with 5% CO2 and N2, whereas other cultures were maintained in 5% CO2 and air in a humidified 37 °C incubator. All experimental treatments comparing normal versus tumour cells were performed in the same RPMI 1640 medium preparations in the 4% O2 chamber until the collection of cells for each assay. For clonogenic survival assays, the complete media and incubator normally used to maintain each cell line were used for the cloning interval. We have also tested the effects of growing HCT116 cells in different pyruvate concentrations (0–1 mM) for 2 weeks or HCT116 and MB231 cells in different O2 partial pressures (4% O2 versus 21% O2) for 4 weeks to determine whether these variables could affect the conclusions of our studies, when comparing these tumour cells with normal cells. The results of these studies (results not shown) indicate that as long as all experimental comparisons are made 24 h after switching the cells being compared to the identical culture conditions, no statistical differences between different maintenance conditions can be detected that would affect the overall conclusions.

Measurement of intracellular superoxide levels

Steady-state levels of superoxide were estimated using oxidation of the fluorescent dye, DHE (dihydroethidine), obtained from Molecular Probes (Eugene, OR, U.S.A.). Cells were washed once with PBS and labelled on culture plates at 37 °C for 40 min in PBS (containing 5 mM pyruvate) with DHE (10 μM; in 0.1% DMSO). Culture plates were placed on ice to stop the labelling, trypsinized and resuspended in ice-cold PBS. Samples were analysed using an FACScan flow cytometer (Becton Dickinson Immunocytometry Systems., Mountain View, CA, U.S.A.) (λex=488 nm and λemission=585 nm band-pass filter). The MFI (mean fluorescence intensity) of 10000 cells was analysed in each sample and corrected for autofluorescence from unlabelled cells. The MFI data were normalized to corresponding normal cell levels for each cell type [18,19].

Measurement of intracellular pro-oxidant levels

Steady-state levels of pro-oxidants (presumably hydroperoxides) were determined using the oxidation-sensitive {CDCFH2 [5- (and 6-)carboxy-2′,7′-dichlorodihydrofluorescein diacetate]; 10 μg/ml} and oxidation-insensitive {CDCF [5- (and 6-)carboxy-2′,7′-dichlorofluorescein diacetate]; 10 μg/ml} fluorescent dyes (dissolved in 0.1% DMSO) that were obtained from Molecular Probes. The cells were washed once with PBS and labelled on the culture plates with the fluorescent dyes for 15 min at 37 °C in PBS. At the end of the incubation time, culture plates were placed on ice, trypsinized, resuspended in ice-cold PBS and analysed using an FACScan flow cytometer (λex=488 nm and λem=530 nm band-pass filter). In each replicate experiment, the numbers obtained for MFI of 10000 cells per sample are arbitrary, based on the gain setting of the flow cytometer adjusted to the normal unlabelled cells in that particular experiment. In order to be able to combine the results of replicate experiments that were performed on different days, normalization to the MFI exhibited by the labelled normal cell type in each experiment was done. The MFI from the normal cell type on a given day was used as the denominator and the MFI obtained from each cancer cell type done on that same day was used as the numerator. Using this procedure, data from each experiment were normalized to the corresponding normal cell type and combined for analysis [19,20].

Glucose consumption

About 150000–300000 cells being compared were plated and allowed to grow for 48 h in 4% O2. Cells were given fresh media at zero time. Cells were counted and media samples were obtained at 24 h and analysed for glucose content using a YSI glucometer. Glucose consumption was determined by subtracting glucose content at 24 h point from the 0 h sample. Results were normalized to the number of cells in each culture [19].

Measurement of G6PD (glucose-6-phosphate dehydrogenase) and 6PGD (6-phosphogluconate dehydrogenase) activities

Exponentially growing HCT116 cancer cells and 33Co normal colon fibroblasts in complete media at 4% O2 were washed with PBS and scrape-harvested in PBS at 4 °C. Combined G6PD+6PGD and 6PGD enzyme activities were measured in cell homogenates as described previously [21]. The first reaction used glucose-6-phosphate and 6-phosphogluconate as substrates, and in the second reaction the substrate consisted of only 6-phosphogluconate without glucose 6-phosphate. The change in attenuance associated with the regeneration of NADPH was monitored at 340 nm. G6PD activity was calculated from the differences observed between the first reaction and the second reaction, and errors were estimated using propagation of error theory. Results were normalized to protein content.

NADPH/NADP+ measurement

After 24 h treatment with (+ Glu) or without (– Glu) glucose, HCT116 and MB231 cells were washed with PBS and scrape- harvested in PBS at 4 °C. After centrifugation at 200 g for 5 min, cell pellets were resuspended in extraction buffer containing 0.1 M Tris/HCl (pH 8.0), 0.01 M EDTA and 0.05% (v/v) Triton X-100. The cell suspension was sonicated at a duty cycle of 34% (Sonics Vibracell, VC750) in ice water. The solution was centrifuged at 2300 g for 5 min. The supernatants were collected and analysed immediately for NADPH and NADP+ as described previously [20]. Results were obtained by comparison with a standard curve using genuine NADPH and normalized per mg of cellular protein.

Thiol analysis

Cells were grown to 70–80% confluency on 100 mm dishes. After treatment, cells were washed with ice-cold PBS, scraped into cold PBS and centrifuged at 4 °C for 5 min at 400 g to obtain cell pellets that were then frozen at −80 °C. Pellets were then thawed and homogenized in 50 mM potassium phosphate buffer (pH 7.8) containing 1.34 mM DTPA (diethylenetriaminepenta-acetic acid). All biochemical determinations were normalized to the protein content by the method of Lowry et al. [22]. GSH content was determined by the GSH/GSSG recycling method [20,23] and the yellow colour change was detected spectrophotometrically at 412 nm. To determine the amount of GSSG, 2 μl of 2-vinylpyridine [1:1 (v/v) in 100% ethanol] was added to 30 μl of sample and incubated for 1 h and assayed as described in [20,23].

Measurement of glucose-deprivation-induced cytotoxicity

Cytotoxicity due to glucose deprivation was measured by plating 200000 cells on to a 60 mm culture dish and incubating in 4% O2 at 37 °C. The cells were left for 24 h to recover from trypsin. At zero time, cells were given fresh glucose-free RPMI 1640 medium containing 10% dialysed FBS and gentamicin. Control cultures were treated identically except that 11 mM glucose was added back at zero time. Clonogenic survival was determined as described previously [24] at 24, 48 and 72 h and normalized to the control cultures for each time point [20,24].

Transduction of antioxidant enzymes

Replication-incompetent adenoviral vectors, namely AdCMV Bgl II (AdBglII), AdCMV mitoCAT (AdmitoCAT) and AdCMV MnSOD (AdMnSOD), were purchased from Viraquest (North Liberty, IA, U.S.A.). They were prepared by inserting the gene of interest into the E1 region of an Ad5 E1/particle E3 deleted replication-deficient adenoviral vector. The cDNAs were all under the control of a CMV (cytomegalovirus) promoter. Except for AdmitoCAT [which was prepared by Dr Shawn W. Flanagan (Department of Radiation Oncology, University of Iowa, Iowa City, IA, U.S.A.)], the adenovirus constructs were originally prepared by Dr John Engelhardt (University of Iowa) [25]. The full-length catalase cDNA with the MnSOD mitochondrial leader sequence added to the construct was originally prepared by Dr J. Andres Melendez (Center for Immunology and Microbial Disease, Albany Medical College, Albany, NY, U.S.A.) [26]. Cells were plated on the day before virus administration. The desired amount of viral particles was added with 1.8 ml of medium per 60 mm dish for 24 h, and then the medium was changed to complete medium and left for another 24 h prior to each experiment.

Measurement of antioxidant enzyme activities

Catalase activity was measured on whole-cell homogenates in 50 mM potassium phosphate (pH 7.0). This method measures the exponential disappearance of H2O2 (10 mM) at 240 nm in the presence of cellular homogenates [27]. The equation that is used to fit the exponential decay of H2O2 for 60 s is: Embedded Image where k is the rate constant that is dependent on the catalase activity. The activities were expressed in milli-k (mk) units/mg of protein added to each assay cuvette as described in [27]. MnSOD activity of cell homogenates was determined as described previously [28] using an indirect competitive inhibition of NBT (Nitro Blue Tetrazolium) reduction assay in the presence of cyanide.

Viability assay

Cells were trypsinized and resuspended in the medium. Cell suspension (0.1 ml) was then mixed with 0.1 ml of 0.04% Trypan Blue dye solution in PBS and incubated at 37 °C for 2 min. The number of cells excluding the dye (viable) and cell stained by the dye (non-viable) were counted. Viability was expressed as the number of cells that excluded the dye divided by the total number (at least 100 cells) counted.

Statistical analysis

Data were expressed as means±1 S.D. unless otherwise specified. One-way ANOVA with Tukey's post-hoc test was used to study the differences between three or more means. Two-way ANOVA with Tukey's post-test was done to determine the differences over various time points. For analysis limited to two groups, Student's t test was used. Significance was determined at P<0.05 and 95% confidence interval.

RESULTS

In order to be able to confirm that colon cancer cells used in the present study (HT29, HCT116 and SW480) have higher pentose phosphate pathway activity and higher glucose consumption compared with their normal counterparts (FHC and 33Co), the cell-mediated rate of glucose disappearance from the medium as well as the G6PD and 6PGD activities were measured (Tables 1 and 2). All three colon carcinoma cell lines demonstrated increased levels of glucose consumption (3- to 7-fold) compared with FHC cells (Table 1). In addition, G6PD and 6PGD activities were significantly elevated (3- and 20-fold respectively) in HCT116 colon cancer cells, relative to 33Co normal colon fibroblasts (Table 2). These results clearly showed that colon cancer cells demonstrated increased glucose consumption and increased pentose phosphate pathway activity, relative to normal cells derived from colon tissue.

View this table:
Table 1 Rates of glucose consumption in normal and cancerous colon epithelial cells (n=3)
View this table:
Table 2 G6PD and 6PGD activity in normal and cancerous colon cells (n=3)

Steady-state levels of O2•− were found to be significantly (P<0.05) elevated (10–20-fold) in colon carcinoma cells (HT29, HCT116 and SW480) compared with normal non-transformed colon epithelial cells (FHC) as determined by increased oxidation of DHE (Figure 1A). When the same cells were treated with the ETC blocker Ant A (antimycin A) (known to increase mitochondrial O2•− production), DHE oxidation was significantly increased in all four cell lines (Figure 1A). The fact that the magnitude of increase in DHE oxidation caused by Ant A in the cancer cells (relative to FHC) was at least as great (if not greater) than that seen in the absence of Ant A strongly suggests that the cancer cell mitochondria are producing much greater amounts of O2•− than are the normal cell mitochondria. Independent comparisons using another normal cell type from colon tissue (33Co; colon fibroblasts) also demonstrated (Figure 1B) significantly greater DHE oxidation (3- to 7-fold) in HT29, HCT116 and SW480 colon carcinoma cells (P<0.05). HCT116 cells treated with 100 units/ml PEG–SOD [poly(ethylene glycol)-conjugated CuZn superoxide dismutase] for 2 h prior to and during DHE labelling demonstrated that 75% of the fluorescent signal was inhibitable by SOD, supporting the conclusion that DHE oxidation was truly reflective of steady-state levels of intracellular O2•− (Figure 1C). Overall, these results strongly support the hypothesis that human colon cancer cells demonstrate greater steady-state levels of intracellular O2•−, relative to normal colon epithelial cells and fibroblasts. The results with Ant A also support the hypothesis that tumour cell mitochondria have a greater capacity for producing superoxide, relative to normal epithelial cell mitochondria.

Figure 1 Steady-state levels of superoxide in normal and cancerous colon cells

(A) Increased steady-state levels of superoxide demonstrated by increased DHE oxidation in human colon cancer cells (HT29, HCT116 and SW480) compared with normal human colon epithelial cells (FHC). Cells were plated on to 60 mm dishes, grown for 48 h and then incubated with 10 μM DHE in 2 ml of PBS containing 5 mM pyruvate at 37 °C for 40 min in the presence or absence of 10 μM Ant A. Cells were trypsinized on ice and analysed by flow cytometry. The MFI of 10000 cells was measured. Values are expressed as the ratio of MFI, relative to FHC cells. Error bars represent ±1 S.D. of three to nine treatment dishes done in three separate experiments. (*Significantly different from FHC, DHE only, P<0.05, N=3; #, significantly different from FHC, DHE+Ant A, P<0.05, N=3–9; ε, significantly different from each respective DHE-only group, P<0.05, N=3–9.) (B) Increased steady-state levels of superoxide demonstrated by increased DHE oxidation in human colon cancer cells (HT29, HCT116, and SW480) compared with normal human colon fibroblasts (33Co). Cells were grown and labelled with 10 μM DHE as described above and analysed by flow cytometry. The MFI of 10000 cells was measured. Values are expressed as the ratio of MFI relative to 33Co cells. Error bars represent ±1 S.D. of nine treatment dishes done in three separate experiments. (*Significantly different from 33Co, DHE only, P<0.05, N=9.) (C) HCT116 cells demonstrated PEG–SOD-inhibitable DHE fluorescence. Cells were plated on to 60 mm dishes, grown for 48 h and treated with 100 units/ml PEG–SOD for 2 h prior to and during DHE labelling. Cells were trypsinized on ice and analysed by flow cytometry. Each sampling measured the MFI of 10000 cells and corrected for autofluorescence. Error bars represent ±1 S.D. of three treatment dishes. (*Significantly different from DHE-only group, P<0.05, N=3.)

Intracellular pro-oxidant production (presumably hydroperoxides) was also determined in colon carcinoma versus normal colon cells using the oxidation-sensitive CDCFH2 in the presence and absence of the ETC blocker Ant A. Using this assay in the absence of Ant A, 20- to 30-fold increases in CDCFH2 oxidation were noted in HCT116 and SW480 cells, relative to FHC, and these results were similar to the increases in DHE oxidation seen in the same cell lines (compare Figure 1A with Figure 2A). In addition, when treated with the Ant A, HCT116 and SW480 cells demonstrated 30- to 40-fold increases in CDCFH2 oxidation, relative to FHC (Figure 2A). Interestingly, relative to FHC, Figure 2(A) shows that HT29 cells demonstrated 2- and 4-fold increases in CDCFH2 oxidation in the absence and presence of Ant A, in contrast with the 10-fold increases in DHE oxidation noted in Figure 1(A). However, the magnitude of increase in CDCFH2 oxidation caused by Ant A in all the cancer cells (relative to FHC) was at least as great (if not greater) than that seen in the absence of Ant A, again strongly suggesting that cancer cell mitochondria are capable of producing greater amounts of hydroperoxides than are normal cell mitochondria. As can be seen in Figure 2(B), all three colon carcinoma cell lines also demonstrated significantly higher CDCFH2 oxidation when compared with 33Co normal colon fibroblasts. The differences seen in MFI values for the cancer cell lines between Figures 1(A) and 1(B) (as well as Figures 2A and 2B) are due to the normalization procedure used to express the data comparing the cancer cell lines with the two different normal cell types originally isolated from colon tissue (FHC colon epithelial cells in Figures 1A and 2A and 33Co fibroblasts in Figures 1B and 2B). Since the normalization procedure was done using two different normal cell types (with different MFIs), the fold changes in normalized MFIs for carcinoma cells were different, depending on which normal cell type was being used for comparison (FHC epithelial cells or 33Co fibroblasts). These results show that regardless of which normal cell type from the colon was used for comparison, the cancer cells clearly demonstrated increased oxidation of DHE or CDCFH2, relative to normal cells. When the cells were labelled with oxidation-insensitive analogue of CDCFH2 (CDCF), there was no significant difference in any of the cell types (Figure 2C), confirming that the differences seen in Figure 2(B) were indeed due to changes in steady-state levels of probe oxidation and not changes in probe influx, efflux or ester cleavage. Overall, these results strongly support the hypothesis that human colon cancer cells demonstrate greater steady-state levels of intracellular hydroperoxides, relative to normal colon epithelial cells and fibroblasts.

Figure 2 Intracellular pro-oxidant levels (presumably hydroperoxides) in normal and cancerous colon cells

(A) Human cancer cells (HT29, HCT116 and SW480) demonstrated significantly increased oxidation of CDCFH2 relative to normal human colon epithelial cells (FHC). Cells were plated on to 60 mm dishes, grown for 48 h and then incubated with 10 μg/ml CDCFH2 in 2 ml of PBS at 37 °C for 15 min in the presence or absence of 10 μM Ant A. Cells were trypsinized on ice and analysed by flow cytometry. The MFI of 10000 cells was measured. Values are expressed as the ratio of MFI relative to FHC cells. Error bars represent ±1 S.D. of three to nine treatment dishes done in three separate experiments. (*Significantly different from FHC, CDCFH2 only, P<0.05, N=3; #, significantly different from FHC, CDCFH2+Ant A, P<0.05, N=3–9; ε, significantly different from each respective CDCFH2-only group, P<0.05, N=3–9.). (B) Human colon cancer cells (HT29, HCT116 and SW480) demonstrated significantly increased oxidation of CDCFH2 relative to normal human colon fibroblasts (33Co). Cells were grown and labelled with 10 μg/ml CDCFH2 and analysed by flow cytometry. The MFI of 10000 cells was measured. Values are expressed as the ratio of MFI relative to 33Co cells. Error bars represent ±1 S.D. of nine treatment dishes done in three separate experiments. (*Significantly different from 33Co, CDCFH2 only, P<0.05, N=9.). (C) The oxidation-insensitive probe demonstrated no differences in fluorescence among normal versus cancer cells from colon tissue. Cells were plated on to 60 mm dishes, grown for 48 h and then incubated with 10 μg/ml CDCF in 2 ml of PBS at 37 °C for 15 min. Cells were trypsinized on ice and analysed by flow cytometry. The MFI of 10000 cells was measured. Values are expressed as the ratio of MFI relative to 33Co cells. Error bars represent ±1 S.D. for three treatment dishes per group done on 3 separate days.

In order to extend these observations to another epithelial cell model system, steady-state levels of DHE and CDCFH2 oxidation were determined in MDA-MB231 breast carcinoma cells and normal non-transformed HMECs. Figure 3 shows that MB231 cells had approx. 2-fold increases in DHE and CDCFH2 oxidation (P<0.05), compared with normal HMECs. Again, no differences were noted when the cells were labelled with the oxidation-insensitive probe fluorescence (CDCF, Figure 3). As was seen with colon epithelial cells, these results support the hypothesis that human breast cancer cells also demonstrate increased steady-state levels of intracellular ROS, relative to normal breast epithelial cells.

Figure 3 Human breast cancer cells (MB231) demonstrated significantly increased oxidation of DHE and CDCFH2, relative to normal HMECs

Cells were grown and labelled with 10 μM DHE, 10 μg/ml CDCFH2 or 10 μg/ml CDCF as described in the Materials and methods section and analysed by flow cytometry. The MFI of 10000 cells was measured. Values are expressed as the ratio of MFI relative to HMECs. Error bars represent ±1 S.D. of three treatment dishes done in three separate experiments. (*Significantly different from HMECs, P<0.05, N=3.)

Since we hypothesized that cancer cells utilize glucose more extensively than their normal counterparts to provide reducing equivalents for the detoxification of endogenous ROS, it was logical to investigate the effects of glucose deprivation on cytotoxicity in normal versus cancer cells. When colon (HCT116) and breast (MB231) carcinoma cells were exposed to glucose-free medium, a significant differential susceptibility to time-dependent clonogenic cell killing (Figures 4A and 4B) was clearly demonstrated in both cancer cell types, relative to normal cells (33Co cells and HMECs). Furthermore, NADPH levels were significantly decreased (and virtually undetectable) after 24 h of glucose deprivation in both cancer cell lines (Table 3). In addition, NADP+ levels increased in both cancer cell lines during 24 h of glucose deprivation, but this increase only reached statistical significance in HCT116 colon carcinoma cells (Table 3). These results strongly suggest that depletion of NADPH levels in colon and breast carcinoma cells could contribute to glucose-deprivation-induced cytotoxicity and oxidative stress in cancer cells.

Figure 4 Clonogenic survival of normal versus cancer cells from colon (A) and breast tissues (B) exposed to glucose deprivation

Cells were plated in complete medium, and 24 h later they were given fresh glucose-free medium containing 10% dialysed FBS, non-essential amino acids and gentamicin. Clonogenic survival was determined at 24, 48 and 72 h and normalized to the respective control group at zero time. Error bars represent ±1 S.E.M. of four to six cloning dishes counted from each treatment dish done in three separate experiments (*Significantly different from 33Co for each time point, P<0.05, N=3; #, significantly different from HMECs, for each time point, P<0.05, N=3.)

View this table:
Table 3 NADPH and NADP+ levels in MB231 and HCT116 cells treated with or without glucose for 24 h (n=3)

ND, not detectable (considered as zero for statistical analysis).

To establish further the causal role of ROS in the cytotoxic mechanisms responsible for differential susceptibility to glucose-deprivation-induced cytotoxicity in colon and breast cancer cells, the effects of co-overexpression of MnSOD and mitoCAT were evaluated. When HCT116 and MB231 cells were transduced with both AdMnSOD and AdmitoCAT [50 MOI (multiplicity of infection) each] and treated for 24 h in the presence or absence of glucose, there was a partial but statistically significant (P<0.05) protection of both cell lines from glucose-deprivation-induced cytotoxicity (Figure 5A). Furthermore, Figure 5(B) demonstrates that during glucose deprivation %GSSG (an indicator of oxidative stress) was significantly elevated and this was partially suppressed by co-overexpression of MnSOD and mitoCAT activity (Figures 5B and 5C) in HCT116 and MB231 cells, relative to vector controls. GSH and GSSG levels in 33Co normal colon fibroblasts and in HMEC normal breast epithelial cells were also measured after glucose deprivation treatment under the same conditions as HCT116 and MB231 cells. In 33Co colon fibroblasts, %GSSG values in the presence and absence of glucose were found to be 5.5±1.8 (mean±1 S.D.) and 5.7±0.6 (mean±1 S.D.) respectively (P>0.05, n=3). GSSG levels of HMECs were undetectable in the presence or absence of glucose. Overall, the results presented in Figures 4 and 5 strongly support a causal link between increased steady-state levels of mitochondrial ROS (i.e. of O2•− and H2O2) and differential cancer cell susceptibility to glucose-deprivation-induced cytotoxicity as well as oxidative stress.

Figure 5 Overexpression of MnSOD and mitoCAT in HCT116 and MB231 cells suppressed the cytotoxicity (A) as well as %GSSG (B) seen at 24 h of glucose deprivation

HCT116 and MB231 cells were transiently transduced with 50 MOI of AdMnSOD and 50 MOI of AdmitoCAT (or 100 MOI AdBgl II, control virus) 24 h after plating. The medium was changed 24 h after infection and cells were allowed to recover 24 h in fresh medium. Cells were then treated with glucose-free medium for an additional 24 h and then plated for clonogenic survival. Survival data were normalized to sham-treated cultures. In (A), error bars represent ±1 S.E.M. of at least six cloning dishes counted from each treatment dish taken from two separate experiments. In (B), error bars represent ±1 S.E.M. of three treatment dishes from each group assayed on three different days. (*Significantly different from Bgl II/+ Glu, P<0.05, N=3; #, significantly different from Bgl II/– Glu, P<0.05, N=3.) In (C), MnSOD and catalase activities measured in HCT116 and MB231 cells given the indicated treatments with adenoviral vectors. U, units.

In order to extend these observations to a clinically relevant inhibitor of glucose metabolism (2DG) capable of mimicking glucose deprivation, FHC and HT29 were treated with 20 mM 2DG for 0–72 h. The results in Figures 6(A) and 6(B) show that HT29 cancer cells were significantly more susceptible to 2DG-induced cell killing as assayed by Trypan Blue dye exclusion, relative to FHC. Since mimicking glucose deprivation by using 2DG is a less severe stress on the cells than the complete glucose deprivation, a 48 h treatment with 2DG was used in an experiment to determine whether overexpression of cellular antioxidants could protect cancer cells from 2DG-induced cytotoxicity. Figure 6(C) demonstrates that co-expression of MnSOD and mitoCAT activities using adenoviral vectors (Figure 6D) significantly protected the HT29 from 2DG-induced toxicity at 48 h of exposure. Overall, the results presented in Figure 6 strongly support a causal link between increased steady-state levels of mitochondrial ROS (i.e. of O2•− and H2O2) and differential cancer cell susceptibility to a clinically relevant inhibitor of glucose metabolism.

Figure 6 Overexpression of MnSOD and mitoCAT suppressed the cytotoxicity of 2DG in HT2 cancer cells

Toxicity of 20 mM 2DG in normal (FHC) cells (A) versus cancer (HT29) cells (B). Overexpression of MnSOD and mitoCAT in HT29 cells suppressed cytotoxicity at 48 h of 2DG treatment (C). In (A, B), viability was assessed using the Trypan Blue dye exclusion assay of at least 100 cells from each group. Error bars represent ±1 S.D. for three measurements from each of the two dishes harvested at each time point (A, B). HT29 cells were transiently transduced with 50 MOI of AdMnSOD and 50 MOI of AdmitoCAT (or 100 MOI AdBgl II, control virus) 24 h after plating. The medium was changed 24 h after infection and cells were allowed to recover for 24 h in fresh medium. Cells were then treated with 20 mM 2DG for an additional 48 h. Clonogenic survival data were normalized to empty-vector-treated cultures (C). Error bars represent ±1 S.E.M. of at least six cloning dishes counted from each treatment dish taken from two experiments (*Significantly different from Bgl II/control, P<0.05; #, significantly different from Bgl II/2DG, P<0.05.) In (D), MnSOD and catalase activities were measured in HT29 cells given the indicated treatments with adenoviral vectors. U, units.

DISCUSSION

The results presented in this study allow for a reformulation of the hypothesis regarding the mechanisms underlying the observed abnormalities in cancer cell glucose metabolism collectively known as the ‘Warburg effect’ [2]. According to Warburg [2], cancer cells demonstrate increased aerobic glycolysis due to damage or impairment of their respiratory mechanism leading to an inability to maintain [ATP] during energy metabolism. However, several lines of evidence suggest that increased aerobic glycolysis in cancer cells may not be the result of an inability to maintain [ATP] during mitochondrial energy metabolism [2931]. First, in many cases tumour cells appear to respire at levels comparable with their non-transformed counterparts [29]. Secondly, recent results suggest that tumour cells do not lack the capacity to utilize oxidative phosphorylation to produce ATP when glycolysis is suppressed [30].

While the ability of mitochondria to consume O2 and produce ATP does not appear to be compromised in cancer cells, mitochondrial structure and mitochondrial DNA integrity have been reported to be abnormal in cancer cells [1416,32]. The mitochondria of malignant cells have been shown to exhibit significant histological abnormalities characterized by unusual arrangements of mitochondrial cristae, mitochondrial hypertrophy and fragmentation when compared with normal cells [14,15]. Furthermore, many tumours, including epithelial cancers (i.e. colon, breast, as well as head and neck), have been shown to have high rates of mtDNA (mitochondrial DNA) mutations (relative to normal human tissues), and this has been suggested to lead to increased O2•− and H2O2 production [16,32]. Therefore it appears that while mitochondrial ATP production may not be compromised in cancer cells, mitochondrial O2•− and H2O2 production could be altered in cancer versus normal cells. Finally, extensive studies using several different cancer cell lines revealed that inhibition of ATP production did not correlate with 2DG-induced radiosensitization, but the extent that glucose uptake and metabolism was increased in tumour cells was an important factor predicting the extent of 2DG-induced radio- and chemo-sensitization [33,34]. While these results seem counter-intuitive to the hypothesis that deficits in energy metabolism are the basis for the ‘Warburg effect’, they are consistent with the hypothesis that increased glucose metabolism in cancer cells could be compensating for elevated steady-state levels of intracellular ROS resulting from defects in mitochondrial respiration that lead to O2•− and H2O2 production.

To the best of our knowledge, this is the first study clearly showing that cancerous human colon and breast epithelial cells have elevated steady-state levels of O2•− and ROS (relative to normal epithelial cells; Figures 1–3) that are accentuated by treatment with an ETC blocker (Ant A; Figures 1 and 2) known to cause increases in mitochondrial O2•− and H2O2 [6,8]. The fact that cancer cells demonstrated profound increases in O2•− and pro-oxidant production (presumably hydroperoxides) when treated with Ant A strongly suggests that one-electron reductions of O2 from ETC complexes I, II and III are more favourable in colon cancer cells, relative to normal cells (Figures 1 and 2). This phenomenon could be explained by (i) defects in mitochondrial respiratory chain assembly and/or (ii) mutations in genes coding for ETC proteins in cancer versus normal cells that increase residence time and/or accessibility of electrons to sites favourable for one-electron reductions of O2 to form O2•− and the resulting dismutation product, H2O2. In this scenario tumour cells, relative to normal cells, would be expected to increase glucose metabolism in order to obtain reducing equivalents such as NADPH via pentose cycle as well as pyruvate from glycolysis [1013] to detoxify H2O2 and other hydroperoxides produced by this defect in respiration.

In strong support of this hypothesis, the data in Figures 4–6 clearly show that cancer cells are more susceptible to killing induced by glucose deprivation or 2DG via a mechanism that can be inhibited by specific scavengers of O2•− and H2O2 targeted to the mitochondria that also inhibited parameters indicative of oxidative stress. These results are consistent with the hypothesis that, relative to normal cells, cancer cells have higher steady-state levels of ROS that are compensated for by increased metabolism of glucose and that when glucose metabolism is restricted, O2•− and H2O2 significantly contribute to the differential susceptibility of cancer cells to oxidative stress and cell killing. This hypothesis is also consistent with previous reports describing biochemical changes consistent with oxidative stress associated with differential susceptibility of transformed versus normal cells to glucose-deprivation-induced cytotoxicity [10,12,13,35].

A mechanistic understanding of the fundamental relationship between glucose metabolism and ROS production in normal versus cancer cells could provide a clear biochemical rationale for the development of combined modality cancer therapies based on inhibiting glucose and hydroperoxide metabolism. Using this rationale it would be predicted that compromising glucose and hydroperoxide metabolism would lead to much greater endogenous metabolic oxidative stress in cancer cells relative to normal cells, which could be exploited to sensitize cancer cells selectively to conventional therapies (i.e. radiation and chemotherapy) that induce oxidative stress while causing minimal normal tissue damage. In support of this idea, previous studies have shown that 2DG selectively radiosensitizes fully transformed rodent cells to a greater extent than non-transformed cells by a mechanism that can be inhibited using the thiol antioxidant, N-acetylcysteine [13]. Furthermore, in MDA-MB231 breast and FaDu human head and neck cancer cells, 2DG-induced cytotoxicity can be significantly enhanced when combined with BSO [L-buthionine-(S,R)-sulfoximine], an inhibitor of GSH synthesis and hydroperoxide metabolism [36,37]. In addition, 2DG sensitizes cancer cells to cisplatin and adriamycin [34,36,38], which are thought to induce oxidative stress. Finally, it has recently been shown that 2DG+cisplatin+radiation is a very effective combination in the FaDu human cancer xenograft model, and 2DG+radiation is an effective combination in a human xenograft model of pancreatic cancer [34,39]. Overall, these results support the hypothesis that a clear mechanistic understanding of the relationship between glucose metabolism and oxidative metabolic defects in cancer versus normal cells can provide a new paradigm useful for the design of combined modality therapies that are selectively cytotoxic to cancer cells.

FUNDING

This work was supported by the NIH (National Institutes of Health; Bethesda, MD, U.S.A.) (grant numbers R01-CA100045, P42-ES013661, R01-CA115438, P30-CA086862 and F32-CA110611).

Acknowledgments

We thank Dr Mary Hendrix for providing MB231 human breast carcinoma cells, and Dr Andre Melendez for the mitoCAT cDNA construct. We also thank Justin Fishbaugh and Gene Hess for their technical assistance with flow cytometry.

Footnotes

  • 1 Dr Larry Oberley sadly died on 21 April 2008, and this paper is dedicated to his memory. He was a great mentor and friend, as well as an originator of the Free Radical Theory of Cancer. He also inspired our present studies with his 1980 paper on mitochondrial abnormalities in cancer cells [Bize, Oberley and Morris (1980) Cancer Res. 40, 3686–3693]. His wisdom and perseverance were an inspiration to all who knew him.

Abbreviations: Ant, A, antimycin A; BSO, L-buthionine-(S,R)-sulfoximine; CDCF, 5- (and 6-)carboxy-2′,7′-dichlorofluorescein diacetate; CDCFH2, 5- (and 6-)carboxy-2′,7′-dichlorodihydrofluorescein diacetate; 2DG, 2-deoxyglucose; DHE, dihydroethidine; ETC, electron transport chain; FBS, fetal bovine serum; G6PD, glucose-6-phosphate dehydrogenase; HMEC, human mammary epithelial cell; MFI, mean fluorescence intensity; mitoCAT, mitochondrially targeted catalase; SOD, superoxide dismutase; MnSOD, Mn-containing SOD; MOI, multiplicity of infection; NBT, Nitro Blue Tetrazolium; O2•−, superoxide radical; PEG–SOD, poly(ethylene glycol)-conjugated CuZn SOD; 6PGD, 6-phosphogluconate dehydrogenase; ROS, reactive oxygen species

References

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