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[Cancer Research 65, 7023-7030, August 1, 2005]
© 2005 American Association for Cancer Research

Epidemiology and Prevention

2-Deoxyglucose as an Energy Restriction Mimetic Agent: Effects on Mammary Carcinogenesis and on Mammary Tumor Cell Growth In vitro

Zongjian Zhu, Weiqin Jiang, John N. McGinley and Henry J. Thompson

Cancer Prevention Laboratory, Colorado State University, Fort Collins, Colorado

Requests for reprints: Henry J. Thompson, Cancer Prevention Laboratory, Colorado State University, 1173 Campus Delivery, Fort Collins, CO 80523. Phone: 970-491-7748; Fax: 970-491-3542; E-mail: henry.thompson{at}colostate.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Dietary energy restriction (DER) is a potent inhibitor of carcinogenesis, but chronic DER in human populations is difficult to sustain. Consequently, interest exists in identifying energy restriction mimetic agents (ERMAs), agents that provide the health benefits of DER without reducing caloric intake. The selection of a candidate ERMAs for this study was based on evidence that DER inhibits carcinogenesis by limiting glucose availability. The study objective was to determine if 2-deoxyglucose (2-DG), a glucose analogue that blocks its metabolism, would inhibit mammary carcinogenesis. Pilot studies were done to establish a dietary concentration of 2-DG that would not affect growth. For the carcinogenesis study, ninety 21-day-old female Sprague-Dawley rats were injected i.p. with 50 mg of 1-methyl-1-nitrosourea per kilogram of body weight. Following injection, animals were ad libitum fed AIN-93G diet containing 0.00%, 0.02%, or 0.03% (w/w) 2-DG for 5 weeks. 2-DG decreased the incidence and multiplicity of mammary carcinomas and prolonged cancer latency (P < 0.05). The 0.02% dose of 2-DG had no effect on circulating levels of glucose, insulin, insulin-like growth factor-I, IGF binding protein-3, leptin, or body weight gain. Using MCF-7 human breast cancer cells to investigate the signaling pathways perturbed by disruption of glucose metabolism, 2-DG reduced cell growth and intracellular ATP in a dose- and time-dependent manner (P < 0.01). Treatment with 2-DG increased levels of phosphorylated AMP-activated protein kinase and Sirt-1 and reduced phosphorylated Akt (P < 0.05). These studies support the hypothesis that DER inhibits carcinogenesis, in part, by limiting glucose availability and that energy metabolism is a target for the development of ERMA for chemoprevention.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
Emerging evidence from studies of human population indicates that excessive caloric intake is associated with an increased risk for cancer and that dietary energy restriction (DER) is protective (1, 2). Similarly, DER has been shown to be a potent inhibitor of carcinogenesis in most of the experimental models in which it has been investigated (3–7). However, current trends in the occurrence of overweight and obesity underscore the difficulty that limiting caloric intake poses to the majority of individuals. This situation gives rise to an important question: "Can agents be identified that mimic the cancer preventive activity of DER in the absence of limiting caloric intake?" This paper presents our initial efforts to identify energy restriction mimetic agents (ERMAs) that inhibit the development of experimentally induced breast cancer.

Relatively little is known about the mechanisms that account for protection against cancer that is provided by DER. However, as summarized in refs. (8, 9), recent evidence from a number of laboratories indicates that DER may mediate its effects by modulating circulating levels of hormones and growth factors. Two classes of molecules that have received attention in this regard are insulin-like growth factors (IGF) and adrenal cortical steroids. Nonetheless, in two recently published proof-in-principle experiments, it was observed that alterations in the metabolism of IGF-I or adrenal cortical steroids were not alone sufficient to account for the protective effects of DER against mammary carcinogenesis (10, 11), although it is likely that changes in both factors contribute to cancer inhibition. Based on these findings, we have formulated an alternative hypothesis of DER-mediated cancer inhibition. We hypothesize that DER imposes a local limitation on glucose availability, selectively, in developing clones of transformed cells due to alterations in glycolysis induced by oncogene activation and/or loss of tumor suppressor gene function (12, 13). In affected transformed cells, reduced glucose availability alters the energy charge of the cell and this in turn modulates the activity of cell signaling pathways that regulate the carcinogenic process. This hypothesis formed the basis for the selection of 2-deoxyglucose (2-DG) as a candidate ERMA for cancer chemoprevention.

2-DG is a widely studied glucose analogue that acts as a competitive inhibitor of glucose metabolism (14). Upon transport into the cell, 2-DG is phosphorylated by hexokinase. However, unlike glucose, 2-DG-PO4 cannot be metabolized by phosphohexose isomerase, which converts glucose-6-phosphate to fructose-6-phosphate (15). Due to low levels of intracellular phosphatase, 2-DG-PO4 is trapped in the cell and is unable to undergo further metabolism. This results in inhibition of glucose metabolism and a reduction in the energy charge of the cell (14). We hypothesized that 2-DG would be taken up to a greater extent by developing clones of transformed cells and, thus, it would be possible to inhibit the carcinogenic process in the absence of an effect on the growth rate of an animal. In this paper, the effects of 2-DG on the postinitiation phase of mammary carcinogenesis were first investigated. We then proceeded to study the effects of 2-DG on cell growth, intracellular concentrations of ATP, and three molecules—AMP-activated protein kinase (AMPK), Akt, and Sirt-1—that are components of pathways that may be regulated by DER (16–19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Chemicals. Primary antibodies used in this study were rabbit anti–phospho-Akt (Ser473), rabbit anti-Akt, rabbit anti-phospho-AMPK (Thr172), rabbit anti-AMPK, anti-rabbit immunoglobulin-horseradish peroxidase-conjugated secondary antibody and LumiGLO reagent with peroxide, all from Cell Signaling Technology (Beverly, MA). Mouse monoclonal anti-Sirt-1 was purchased from Upstate Cell Signaling Solution (Lake Placid, NY). Mouse anti–ß-actin primary antibody, 2-DG, and crystal violet were obtained from Sigma Chemical Co. (St. Louis, MO). 1-Methyl-1-nitrosourea was obtained from Ash Stevens (Detroit, MI). Anti-mouse immunoglobulin-horseradish peroxidase–conjugated secondary antibody was purchased from Santa Cruz Corporation (Santa Cruz, CA). DMEM and fetal bovine serum were purchased from Invitrogen Corp. (Carlsbad, CA). Human breast cancer MCF-7 cell line was obtained from American Type Culture Collection (Manassas, VA).

Dose-finding studies. Two experiments were conducted to determine a dietary concentration of 2-DG that could be fed without affecting growth rate. Female Sprague-Dawley rats obtained from Taconic Farms (Germantown, NY) at 24 days of age were used for these experiments. The first study was based on published data indicating that concentrations between 0.2% and 0.4% (w/w) were well tolerated by the rat (20). Three days after receipt, 24 animals were randomized to one of three groups and were fed a published modification of AIN-93G (21) or that diet supplemented with 0.2% or 0.4% 2-DG. These treatments were terminated after 1 week of feeding because of a significant inhibitory effect on growth rate. For the second dose-finding study, 36 female Sprague-Dawley rats were obtained and were randomized into six groups fed ad libitum AIN-93G diet containing 0.0%, 0.02%, 0.04%, 0.06%, 0.08%, or 0.10% (w/w) 2-DG for 6 weeks. Animals were weighed daily.

Carcinogenesis study. For the carcinogenesis study, 90 female Sprague-Dawley rats were obtained from Taconic Farms at 20 days of age. At 21 days of age, rats were injected with 50 mg 1-methyl-1-nitrosourea per kilogram of body weight (i.p.) as previously described (22). Rats were housed two per cage in solid-bottomed polycarbonate cages equipped with a food cup. Six days following carcinogen injection, all rats were randomized into one of three groups, 30 rats per group, and were fed ad libitum AIN-93G diet containing 0.0%, 0.02%, or 0.03% (w/w) 2-DG for 5 weeks. Animal rooms were maintained at 22 ± 1°C with 50% relative humidity and a 12-hour light/12-hour dark cycle. Rats were weighed thrice per week and were palpated for detection of mammary tumors twice per week starting from 19 days postcarcinogen. The work reported was reviewed and approved by the Institutional Animal Care and Use Committee and conducted according to the committee guidelines.

Necropsy. Following an overnight fast, rats were killed over a 3-hour time interval via inhalation of gaseous carbon dioxide. The sequence in which rats were euthanized was stratified across groups so as to minimize the likelihood that order effects would masquerade as treatment associated effects. After the rats lost consciousness, blood was directly obtained from the retro-orbital sinus and gravity fed through heparinized capillary tubes (Fisher Scientific, Pittsburgh, PA) into EDTA coated tubes (Becton Dickinson, Franklin Lakes, NJ) for plasma or plastic tubes for serum. The bleeding procedure took ~1 minute per rat. Plasma or serum was isolated by centrifugation at 1,000 x g for 10 minutes at room temperature.

For the carcinogenesis study, following blood collection and cervical dislocation, rats were then skinned and the skin was examined under translucent light for detectable mammary pathologies. All grossly detectable mammary gland lesions were excised. In addition, whole mounts of abdominal-inguinal mammary gland chains were prepared and tissue was fixed in methacarn. The fixed tissues were subsequently stained, and stained whole mounts were photographed and then evaluated under x2 magnification for detection of any abnormality that might be a mammary pathology as previously described in detail (22). All lesions and abnormalities were processed for histologic classification as described in ref. (23). The following pathologies were observed: intraductal proliferations, ductal carcinoma in situ, and adenocarcinomas.

Assessment of circulating molecules. Whole blood glucose was determined using ONETOUCH Ultra Test Strips (Lifescan, Inc., Milpitas, CA). Serum insulin was determined by a commercial rat/mouse insulin ELISA kit (Linco Research, Inc., Charles, Missouri) using rat insulin for the preparation of the standards in the assay. Plasma insulin-like growth factor-I (IGF-I) was determined using a commercial rat IGF-I enzyme immunoassay kit (Diagnostic Systems Laboratories, Inc., Webster, TX). Serum corticosterone was determined by a commercial corticosterone enzyme immunoassay kit (Diagnostic Systems Laboratories) using the rat corticosterone standards and controls in the assay. Plasma leptin was determined by a commercial murine leptin enzyme immunoassay kit (Diagnostic Systems Laboratories).

Serum IGF binding protein-3 (IGFBP-3) was determined by Western ligand blotting using biotinyl IGF-I (GroPep Limited, Adelaide, SA, Australia) as previously reported by our laboratory (10). Briefly, 1.5 µL serum were run on a 12% Tris-glycine gel (Invitrogen Life Technologies, Carlsbad, CA) using the XCell Mini-Cell system (Novex Electrophoresis, San Diego, CA) under nondenaturing and nonreducing conditions. For ligand blots, proteins were transferred to a nitrocellulose membrane. Membranes were washed with TBS containing 1% (v/v) Tween 20 for 30 minutes at room temperature and preblocked with 1% (v/v) bovine serum albumin in TBS containing 0.1% (v/v) Tween 20 overnight before incubation with 2 µg of biotinyl–IGF-I (GroPep) in TBS for 90 minutes at room temperature. After sequential washes in TBS containing 0.1% (v/v) Tween 20, membranes were incubated in 1:1,000 dilution of avidin-horseradish peroxidase [in TBS-0.1% (v/v) Tween 20] for 45 minutes at room temperature. After developing the membrane with LumiGLO reagent, the chemiluminescence signal was captured using ChemiDoc (Bio-Rad, Hercules, CA) and Quantity One software (Bio-Rad) was used to quantify the signal intensity of the bands.

Cell culture and whole cell extract preparation. MCF-7 cells, a human breast cancer cell line, were cultured in DMEM medium, supplemented with 10% fetal bovine serum. Cells were maintained in 5% CO2 atmosphere at 37°C.

Cell growth assay. The effect of 2-DG on cell growth was determined by evaluating the number of adherent cells as described previously (24). Briefly, MCF-7 cells were plated at 3 x 104 cells per well in flat-bottomed 96-well plates in 100 µL of culture medium under the culture conditions detailed above. After 24 hours, cells were fed with fresh medium including 2-DG at doses of 0, 4, 8, or 16 mmol/L. At days 1, 3, and 5 after 2-DG exposure, cells were fixed with 1% glutaraldehyde, replaced with PBS, and stored at 4°C. At the end of an experiment, all of the plates were stained with 0.02% aqueous crystal violet for 30 minutes and rinsed with deionized water. After redissolving the bound crystal violet in 70% ethanol, the absorbance was determined at 590 nm using a SPECTRA MAX PLUS Microplate Spectrophotometer System (Molecular Devices, Sunnyvale, CA).

ATP assay. The effect of 2-DG on ATP level in the cells was determined using an ENLITEN ATP assay kit (Promega Corporation, Kadison, WI), the bioluminescence was detected using a TD 20/20 luminometer (Turner Biosystem, Sunnyvale, CA), and the amount of ATP per well was standardized by the cell number estimated by crystal violet method described above.

Western blotting. Logarithmically growing semiconfluent cultures of MCF-7 cells were treated with 0, 4, 8, or 16 mmol/L dose of 2-DG for 1, 3, and 5 days. The medium was aspirated at the end of these treatments, and the cells were quickly washed twice with ice-cold PBS. A 0.1 mL aliquot of lysis buffer [20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium PPi, 1 mmol/L ß-glycerolphosphate, 1 mmol/L Na3VO4, 1 µg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride] was then added per 60-mm-diameter dish. After bathing in lysis buffer for 15 minutes on ice, the cells were scraped from the dish; the mixture of buffer and cells was transferred to microfuge tubes and left in ice for an additional 15 minutes. The lysates were collected by centrifugation for 15 minutes in a tabletop centrifuge at 4°C, and protein concentration in the clear supernatants was determined by the Bio-Rad protein assay.

Western blotting was done as described before (19). Briefly, 40 µg of protein lysate per sample were denatured with SDS sample buffer [63 mmol/L Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mmol/L DTT, and 0.01% bromophenol blue], subjected to SDS-PAGE on 12% gel, and the protein bands blotted onto a membrane. The levels of phospho-Akt (Ser473), Akt, phospho-AMPK (Thr172), AMPK, and Sirt-1 were determined using specific primary antibodies, followed by treatment with the appropriate peroxidase-conjugated secondary antibody and visualized by LumiGLO reagent Western blotting detection system. The chemiluminescence signal was captured using ChemiDoc (Bio-Rad) and Quantity One software (Bio-Rad) was used to quantify the signal intensity of the bands.

Statistical analyses. Differences among groups in the incidence of premalignant and malignant mammary gland pathologies were evaluated by {chi}2 analysis (25). Differences among groups in the number of premalignant and malignant pathologies per rat (multiplicity) were evaluated by ANOVA after square root transformation as recommended in ref. (26). Cancer latency was evaluated by the procedure of Mantel (27). Final body weights and circulating levels of glucose, insulin, corticosterone, leptin, and IGF-I were analyzed by ANOVA (27). Pairwise comparisons among treatments were evaluated using the method recommended by Fischer (27).

Differences in the number of MCF-7 cells and ATP level of the cells following exposure to 2-DG at different doses and time points were evaluated by factorial ANOVA of the absorbance data resulting from each assay although the data are shown normalized to the control to facilitate graphical presentation and visual interpretation (27). Data derived from Western ligand blot and Western blot analyses represent semiquantitative estimates of the amount of a specific protein that is present in serum or a cell extract. This fact was taken into account in the statistical evaluation of the data. The data displayed in the graphs are reported as means of the ratio (experimental/control) of the actual scanning units derived from the densitometric analysis of each Western blot. All values are the means of three independent experiments. For statistical analyses, the actual scanning density data derived from the analysis of the Western blots using Quantity One (Bio-Rad) were first ranked. This approach is particularly suitable for semiquantitative measurements that are collected as continuously distributed data. This approach has the advantage of maintaining the relative relationships among data being compared without giving undue weight to outlying results. The ranked data were then subjected to factorial ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
Dose-Finding Studies
First study. The effect of 0.2% and 0.4% (w/w) 2-DG on body weight was compared with that of animals fed AIN-93G alone. Within 4 days of initiating dietary treatment, growth inhibition by 2-DG was observed and by day 7, a difference of >20% existed between animals fed AIN-93G (108 ± 2 g) and those fed 0.2% (77 ± 2 g) or 0.4% (78 ± 3 g) 2-DG. Consequently, the study was terminated because the goal was to identify a dose of 2-DG that did not affect the rate of growth.

Second study. In this study, rats were fed diets containing 2-DG at 0%, 0.02%, 0.04%, 0.06%, 0.08%, and 0.10% (w/w) for 45 days to determine a dose of 2-DG that did not have an effect on body weight gain. As shown in Fig. 1, 0.02% 2-DG had no effect on body weight gain. A dose of 0.04% in the diet had a small inhibitory effect on growth, but the reduction was not statistically significant (Table 1). The body weight of rats fed 2-DG at 0.06% to 0.10% in the diet were significantly reduced compared with the control rats (P < 0.01). Fasting blood glucose concentrations of rats fed 2-DG were determined at the end of the study. Compared to the control rats, levels of glucose were reduced at doses of 2-DG above 0.04% in the diet (P = 0.03). Based on these results, doses of 0.02% and 0.03% 2-DG were selected for the carcinogenesis study.



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  		Figure 1. Effect of feeding five different concentrations of 2-DG in the diet on the body weight gain of female Sprague-Dawley rats. The control group (0.00% 2-DG) was fed AIN-93G as described in Materials and Methods. Only 0.02% 2-DG did not affect the rate of growth.

 

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  		Table 1. Final body weights and fasting blood glucose of rats fed 2-DG in diet

 
Carcinogenic Response
The incidence of both premalignant and malignant mammary gland pathologies was significantly reduced by 2-DG as shown in Table 2 and Fig. 2 and cancer latency was delayed (P < 0.001). 2-DG also significantly reduced the multiplicity of mammary carcinomas compared with control rats (P < 0.05). The number of premalignant mammary pathologies was also decreased in rats fed 2-DG compared with the control rats, but the differences were not statistically significant. Final body weights of rats fed 0.02% and 0.03% 2-DG were 2% (P > 0.05) and 7% (P < 0.05) lower, respectively, than those of control rats. The difference between the control group and rats fed 0.03% 2-DG became significant statistically only during the final week of the study.


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  		Table 2. Effects of 2-DG on the carcinogenic response in the mammary gland

 


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  		Figure 2. Effect of feeding either 0.02% or 0.03% (w/w) 2-DG on rate of growth (A), the incidence of palpable mammary tumors that were histopathologically confirmed to be mammary carcinomas (B), and the average number of histopathologically confirmed palpable mammary cancers per rat as a function of time post carcinogen. Animals fed 0.02% 2-DG did not differ in growth from animals fed the control diet (0.00% 2-DG), whereas 0.03% 2-DG caused a 7% decrease in final weight that was statistically significant (P < 0.05). Treatment with 2-DG significantly delayed the appearance of palpable mammary carcinomas (P < 0.001).

 
Circulating Factors
As shown in Table 3, compared with control rats, the concentration of insulin (P < 0.001) in serum was significantly reduced by 0.03% 2-DG, but not 0.02% 2-DG. Serum corticosterone was elevated by both concentrations of 2-DG (P < 0.001). No significant differences were observed in the level of serum leptin, plasma IGF-I, or IGFBP-3, although a reduction in the concentration of the binding protein with increasing concentration of 2-DG approached statistical significance (P = 0.06).


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  		Table 3. Effect of 2-DG on circulating concentrations of growth factors and hormones

 
Effects of 2-Deoxyglucose on Cell Growth and ATP Level
Table 4 and Fig. 3A and B show the effects of increasing concentrations of 2-DG on growth of cultured MCF-7 human breast cancer cells. Cell growth was significantly inhibited by 2-DG at doses of 4, 8, or 16 mmol/L for 1, 3, or 5 days in a dose- and time-dependent manner (P < 0.001). The effects of 2-DG on the cellular concentration of ATP in MCF-7 are shown in Fig. 3C and D. Exposure to 2-DG at 4, 8, or 16 mmol/L for 1, 3, or 5 days significantly reduced the level of ATP in MCF-7 cells in a dose- and time-dependent manner (P < 0.001) that paralleled the effects of 2-DG on cell growth.


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  		Table 4. Effects of 2-DG on growth of MCF-7 human breast cancer cells

 


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  		Figure 3. Dose- and time-dependent effect of 2-DG on cell growth and intracellular levels of ATP in MCF-7 human breast cancer cells. MCF-7 cells were exposed to 2-DG at 0, 4, 8, or 16 mmol/L for 1, 3, and 5 days as detailed in Materials and Methods. All experiments were repeated thrice. In each experiment, eight replicates at each concentration per time point were analyzed. The results of a representative experiment are presented. Points, means for both cell growth (A and B) and ATP concentration (C and D) expressed as a percent of values observed in controls to facilitate visual interpretation of the data; bars, SE. However, statistical analyses were done on the raw absorbance data via factorial ANOVA. These analyses showed a 2-DG dose- and time-dependent reduction in cell growth (P < 0.01) and ATP concentration (P < 0.01), and that there was a significant interaction between dose of 2-DG and duration of exposure for both cell growth (P < 0.01) and ATP concentration (P < 0.04).

 
Effects of 2-Deoxyglucose on Protein Levels of Energy Metabolism Regulatory Molecules
As shown in Fig. 4, the levels of phosphorylated Akt were significantly decreased, whereas the levels of phosphorylated AMPK and Sirt-1 were significantly increased in MCF-7 cells exposed to 2-DG at 4, 8, or 16 mmol/L for 1, 3, or 5 days in a dose- and time-dependent manner (P < 0.05).



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  		Figure 4. Effect of 2-DG exposure on the expression of the following proteins: phosphorylated Akt, Akt, phosphorylated AMPK, AMPK, and Sirt-1. ß-actin expression was used as the internal control. MCF-7 human breast cancer cells were exposed to 0, 4, 8, or 16 mmol/L 2-DG for 1, 3, and 5 days as detailed in Materials and Methods. All experiments were repeated thrice. In each experiment, three replicates at each concentration per time point were analyzed. The results of a representative experiment are presented. The ratios were calculated as the absorbance of a specific protein from treated cells divided by the absorbance for that protein in control cells normalized to the expression of ß-actin. The results of a representative experiment are presented. Statistical analyses were done on the ranks of the raw absorbance data via factorial ANOVA as described in Materials and Methods. These analyses showed a 2-DG dose- and time-dependent reduction in levels of phospho-Akt and an increase in the levels of phospho-AMPK and Sirt-1 (P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
Because of the accumulating evidence of the tremendous health burden represented by the prevalence of overweight and obesity that has been reported globally, and an extensive base of experimental data that indicate that DER is protective against a number of chronic disease including cancer (1, 3–7), there is an emerging interest in identifying agents that provide the health benefits of DER in the absence of restricting caloric intake (15). We refer to such agents as ERMA. There have been a limited number of reports on the investigation of ERMA for the delay of aging and in the treatment of diabetes (20, 28–30), but to our knowledge this is the first report on the investigation of ERMA for cancer chemoprevention. We expect that at least initially, the goals of investigators seeking to identify ERMA will vary depending on the disease process for which ERMA are being investigated and that even within disease process, the criteria for selecting ERMA will differ. In the experiments reported in this study, our selection of an ERMA was based on the hypothesis that the intracellular limitation of glucose availability would inhibit the carcinogenic process. Accordingly, the criteria for dose selection of 2-DG were very restrictive so that we could not only evaluate the cancer inhibitory activity of 2-DG, but also test the underlying hypothesis. Thus, we sought to identify a dietary concentration of 2-DG that could be fed continuously without appreciably affecting the rate of body weight gain, circulating levels of blood glucose, or levels of hormones or growth factors that DER modulates and that have been implicated in accounting for the cancer inhibitory activity of DER (8, 9). Based on data from our dose-finding studies (Table 1; Fig. 1), we selected for the carcinogenesis study the highest two concentrations of 2-DG that we thought would fulfill these criteria. The data shown in Table 2 and Fig. 2 indicate that a concentration of 0.02% 2-DG largely fulfilled these criteria, whereas 0.03% 2-DG had some effect on weight gain and circulating factors. However, no gross evidence of toxicity of any type was noted at either dose of 2-DG, an observation consistent with chronic feeding studies in rats in which much higher doses of 2-DG have been reported to be well tolerated without toxicity (31).

As shown in Fig. 2B and C and summarized in Table 2, the incidence and multiplicity of mammary carcinomas was significantly reduced by both 0.02% and 0.03% 2-DG and cancer latency was prolonged. The magnitude of these effects is similar to that observed in response 20% DER in the same model system (21); however, in the case of the 0.02% dose of 2-DG, the inhibition of carcinogenesis was observed in the absence of effects of body weight, blood glucose, or changes in insulin, IGF-I, IGFBP-3, or leptin and only a 20% elevation in plasma corticosterone. The incidence of premalignant pathologies (intraductal proliferation and ductal carcinoma in situ) was also significantly reduced by both doses of 2-DG and the average number of these pathologies per rat was also reduced, but that reduction was not statistically significant. Collectively, these data suggest that perturbing intracellular metabolism of glucose acts at an early stage in the disease process as would be predicted based on evidence that energy production in transformed cells switches primarily to glycolysis, the Warberg effect, and that this switch is induced by oncogene activation and/or loss of tumor suppressor gene function (12, 13, 32).

Whereas the evidence presented above is supportive of our working hypothesis that limitation of glucose availability can inhibit the development of cancer and that this mechanism may account, in part, for the cancer preventive activity of DER, we considered it important to investigate the cellular and molecular effects of 2-DG in a highly controlled model system that is relevant to breast cancer. For this work, we chose the widely studied MCF-7 human breast cancer cell line. The first goal of this work was to determine (a) if 2-DG could inhibit cell number accumulation, an effect predicted by its in vivo inhibition of carcinogenesis and (b) whether cellular energy charge would be reduced. As shown in Table 4, 2-DG induced a dose- and time-dependent reduction in cell growth, and the data in Fig. 4 show that ATP levels were reduced by 2-DG in a dose-dependent (P < 0.05) and time-dependent (P < 0.05) manner that paralleled its effects on cell growth. The effect on cell growth is consistent with 2-DG exerting a specific effect on cell proliferation and/or apoptosis, which are involved in the loss of regulation of tissue size homeostasis that results in tumor occurrence (33). The effects of 2-DG on these cellular processes are currently under investigation.

The second goal of the in vitro work was to test three straightforward predictions. First, based on evidence reviewed in ref. (34), we predicted that the reduction in cellular energy charge induced by 2-DG would result in the activation of AMPK. As shown in Fig. 4, levels of phospho-AMPK were clearly elevated in a dose- and time-dependent manner. This finding has two implications for future in vivo investigations. The work in this study was based on the hypothesis that DER induces a local limitation in glucose availability resulting in a reduced cellular energy charge that preferentially affects transformed cells because of their differential utilization of glucose relative to their nontransformed counterparts. Based on this, we predicted that 2-DG would preferentially accumulate in these cells and inhibit their growth. However, to test this hypothesis in vivo at the level of intracellular glucose or ATP concentrations in only transformed cells is difficult from a technical perspective, although the work of Sauer and Dauchy (35) may be helpful in this regard. On the other hand, testing the hypothesis that either DER or 2-DG alters cellular energy charge in developing premalignant and malignant cells using a surrogate marker, such as AMPK activation, is clearly achievable and the present work provides a rationale for pursuing those studies.

The second prediction tested in this cell culture work was that reducing cellular energy charge using 2-DG would emulate the effects of DER on cell signaling as reported in ref. (19), and that the concentration of phospho-Akt would be reduced. The data shown in Fig. 4 are consistent with this prediction and provide a rationale for examining the effects of 2-DG on the signaling pathway of which Akt is a component in vivo under conditions in which circulating levels of IGF-I and IGFBP-3 are not affected. This is critical because it currently is thought that DER is most likely to affect the activity of Akt via its effects on IGF-I signaling through IGF-IR (8). Whereas this expectation is quite logical, it could be that cross-talk occurs between gene products of the pathways of which AMPK and Akt are components and that not all effects are mediated through IGF-IR.

Finally, recently reported work has implicated the sertuin family of genes, and in particular Sirt-1, as being induced by DER and in accounting for some of the health benefits of DER (17, 18). If elevated expression of Sirt-1 is a signature for DER, then we predict that if DER acts by limiting glucose availability, Sirt-1 would be induced by treatment with 2-DG. As shown in Fig. 4, Sirt-1 was induced in a dose- and time-dependent manner by 2-DG. Whether the regulation of Sirt-1 is linked to pathways of which AMPK and/or Akt are components is currently under investigation.


   Conclusion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
A primary goal of the experiments reported in this study was to determine whether a competitive inhibitor of glucose metabolism would mimic the effects of DER by inhibiting the process of experimentally induced mammary carcinogenesis. It was anticipated that these experiments would also inform our working hypothesis that DER inhibits tumor development by limiting glucose availability. The in vivo and in vitro data obtained indicate that under very restrictive conditions, 2-DG does inhibit carcinogenesis without inducing many of the systemic effects that accompany DER. The in vitro data show that 2-DG reduces cellular energy charge and identified three signaling pathways that seem to be modulated by limiting energy availability. These studies provide a basis for further in vivo studies of the mechanisms of cancer inhibition by DER and for in vitro and in vivo experiments that seek to identify ERMA that inhibit the carcinogenic process.


   Acknowledgments
 
Grant support: USPHS grant CA52626 from the National Cancer Institute.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/ 9/05. Revised 4/ 6/05. Accepted 5/18/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

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