2-Deoxyglucose as an Energy Restriction Mimetic Agent: Effects on

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1 Research Article 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 Abstract limiting caloric intake? This paper presents our initial efforts to Dietary energy restriction (DER) is a potent inhibitor of identify energy restriction mimetic agents (ERMAs) that inhibit the carcinogenesis, but chronic DER in human populations is development of experimentally induced breast cancer. difficult to sustain. Consequently, interest exists in identifying Relatively little is known about the mechanisms that account for energy restriction mimetic agents (ERMAs), agents that provide protection against cancer that is provided by DER. However, as the health benefits of DER without reducing caloric intake. summarized in refs. (8, 9), recent evidence from a number of The selection of a candidate ERMAs for this study was based laboratories indicates that DER may mediate its effects by on evidence that DER inhibits carcinogenesis by limiting modulating circulating levels of hormones and growth factors. glucose availability. The study objective was to determine if Two classes of molecules that have received attention in this regard 2-deoxyglucose (2-DG), a glucose analogue that blocks its are insulin-like growth factors (IGF) and adrenal cortical steroids. Nonetheless, in two recently published proof-in-principle experi- metabolism, would inhibit mammary carcinogenesis. Pilot studies were done to establish a dietary concentration of 2-DG ments, it was observed that alterations in the metabolism of IGF-I that would not affect growth. For the carcinogenesis study, or adrenal cortical steroids were not alone sufficient to account ninety 21-day-old female Sprague-Dawley rats were injected for the protective effects of DER against mammary carcinogenesis i.p. with 50 mg of 1-methyl-1-nitrosourea per kilogram of body (10, 11), although it is likely that changes in both factors contribute weight. Following injection, animals were ad libitum fed AIN- to cancer inhibition. Based on these findings, we have formulated 93G diet containing 0.00%, 0.02%, or 0.03% (w/w) 2-DG for an alternative hypothesis of DER-mediated cancer inhibition. We 5 weeks. 2-DG decreased the incidence and multiplicity of hypothesize that DER imposes a local limitation on glucose mammary carcinomas and prolonged cancer latency (P < availability, selectively, in developing clones of transformed cells 0.05). The 0.02% dose of 2-DG had no effect on circulating due to alterations in glycolysis induced by oncogene activation levels of glucose, insulin, insulin-like growth factor-I, IGF and/or loss of tumor suppressor gene function (12, 13). In affected binding protein-3, leptin, or body weight gain. Using MCF-7 transformed cells, reduced glucose availability alters the energy human breast cancer cells to investigate the signaling path- charge of the cell and this in turn modulates the activity of cell signaling pathways that regulate the carcinogenic process. This ways perturbed by disruption of glucose metabolism, 2-DG reduced cell growth and intracellular ATP in a dose- and time- hypothesis formed the basis for the selection of 2-deoxyglucose dependent manner (P < 0.01). Treatment with 2-DG increased (2-DG) as a candidate ERMA for cancer chemoprevention. levels of phosphorylated AMP-activated protein kinase and 2-DG is a widely studied glucose analogue that acts as a Sirt-1 and reduced phosphorylated Akt (P < 0.05). These competitive inhibitor of glucose metabolism (14). Upon transport studies support the hypothesis that DER inhibits carcino- into the cell, 2-DG is phosphorylated by hexokinase. However, genesis, in part, by limiting glucose availability and that unlike glucose, 2-DG-PO4 cannot be metabolized by phospho- energy metabolism is a target for the development of ERMA hexose isomerase, which converts glucose-6-phosphate to fructose- for chemoprevention. (Cancer Res 2005; 65(15): 7023-30) 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 Introduction reduction in the energy charge of the cell (14). We hypothesized Emerging evidence from studies of human population indicates that 2-DG would be taken up to a greater extent by developing that excessive caloric intake is associated with an increased risk for clones of transformed cells and, thus, it would be possible to cancer and that dietary energy restriction (DER) is protective (1, 2). inhibit the carcinogenic process in the absence of an effect on the Similarly, DER has been shown to be a potent inhibitor of growth rate of an animal. In this paper, the effects of 2-DG on the carcinogenesis in most of the experimental models in which it has postinitiation phase of mammary carcinogenesis were first been investigated (37). However, current trends in the occurrence investigated. We then proceeded to study the effects of 2-DG on of overweight and obesity underscore the difficulty that limiting cell growth, intracellular concentrations of ATP, and three caloric intake poses to the majority of individuals. This situation moleculesAMP-activated protein kinase (AMPK), Akt, and Sirt- gives rise to an important question: Can agents be identified that 1that are components of pathways that may be regulated by DER mimic the cancer preventive activity of DER in the absence of (1619). Requests for reprints: Henry J. Thompson, Cancer Prevention Laboratory, Materials and Methods Colorado State University, 1173 Campus Delivery, Fort Collins, CO 80523. Phone: 970-491-7748; Fax: 970-491-3542; E-mail: [email protected] Chemicals. Primary antibodies used in this study were rabbit anti I2005 American Association for Cancer Research. phospho-Akt (Ser473), rabbit anti-Akt, rabbit anti-phospho-AMPK (Thr172), www.aacrjournals.org 7023 Cancer Res 2005; 65: (15). August 1, 2005

2 Cancer Research 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. rabbit anti-AMPK, anti-rabbit immunoglobulin-horseradish peroxidase- body weight (i.p.) as previously described (22). Rats were housed two per conjugated secondary antibody and LumiGLO reagent with peroxide, all cage in solid-bottomed polycarbonate cages equipped with a food cup. Six from Cell Signaling Technology (Beverly, MA). Mouse monoclonal anti-Sirt-1 days following carcinogen injection, all rats were randomized into one of was purchased from Upstate Cell Signaling Solution (Lake Placid, NY). three groups, 30 rats per group, and were fed ad libitum AIN-93G diet Mouse antih-actin primary antibody, 2-DG, and crystal violet were containing 0.0%, 0.02%, or 0.03% (w/w) 2-DG for 5 weeks. Animal rooms obtained from Sigma Chemical Co. (St. Louis, MO). 1-Methyl-1-nitrosourea were maintained at 22 F 1jC with 50% relative humidity and a 12-hour was obtained from Ash Stevens (Detroit, MI). Anti-mouse immunoglobulin- light/12-hour dark cycle. Rats were weighed thrice per week and were horseradish peroxidaseconjugated secondary antibody was purchased palpated for detection of mammary tumors twice per week starting from from Santa Cruz Corporation (Santa Cruz, CA). DMEM and fetal bovine 19 days postcarcinogen. The work reported was reviewed and approved by serum were purchased from Invitrogen Corp. (Carlsbad, CA). Human breast the Institutional Animal Care and Use Committee and conducted according cancer MCF-7 cell line was obtained from American Type Culture to the committee guidelines. Collection (Manassas, VA). Necropsy. Following an overnight fast, rats were killed over a 3-hour Dose-finding studies. Two experiments were conducted to determine a time interval via inhalation of gaseous carbon dioxide. The sequence in dietary concentration of 2-DG that could be fed without affecting growth which rats were euthanized was stratified across groups so as to minimize rate. Female Sprague-Dawley rats obtained from Taconic Farms (German- the likelihood that order effects would masquerade as treatment associated town, NY) at 24 days of age were used for these experiments. The first study effects. After the rats lost consciousness, blood was directly obtained from was based on published data indicating that concentrations between 0.2% the retro-orbital sinus and gravity fed through heparinized capillary tubes and 0.4% (w/w) were well tolerated by the rat (20). Three days after receipt, (Fisher Scientific, Pittsburgh, PA) into EDTA coated tubes (Becton 24 animals were randomized to one of three groups and were fed a published Dickinson, Franklin Lakes, NJ) for plasma or plastic tubes for serum. The modification of AIN-93G (21) or that diet supplemented with 0.2% or 0.4% bleeding procedure took f1 minute per rat. Plasma or serum was isolated 2-DG. These treatments were terminated after 1 week of feeding because of a by centrifugation at 1,000 g for 10 minutes at room temperature. significant inhibitory effect on growth rate. For the second dose-finding For the carcinogenesis study, following blood collection and cervical study, 36 female Sprague-Dawley rats were obtained and were randomized dislocation, rats were then skinned and the skin was examined under into six groups fed ad libitum AIN-93G diet containing 0.0%, 0.02%, 0.04%, translucent light for detectable mammary pathologies. All grossly 0.06%, 0.08%, or 0.10% (w/w) 2-DG for 6 weeks. Animals were weighed daily. detectable mammary gland lesions were excised. In addition, whole Carcinogenesis study. For the carcinogenesis study, 90 female Sprague- mounts of abdominal-inguinal mammary gland chains were prepared and Dawley rats were obtained from Taconic Farms at 20 days of age. At 21 days tissue was fixed in methacarn. The fixed tissues were subsequently stained, of age, rats were injected with 50 mg 1-methyl-1-nitrosourea per kilogram of and stained whole mounts were photographed and then evaluated under Table 1. Final body weights and fasting blood glucose of rats fed 2-DG in diet 2-DG % (w/w) Overall P 0.00 (Control) 0.02 0.04 0.06 0.08 0.10 Final body weight (g) 215 F 7a 216 F 5a 200 F 5a 162 F 11b 152 F 1b 113 F 7c

3 2-Deoxyglucose and Mammary Carcinogenesis Table 2. Effects of 2-DG on the carcinogenic response in the mammary gland Control (n = 30) 0.02% 2-DG (n = 30) 0.03% 2-DG (n = 30) Overall P Incidence of AC (%) 83.3 (25)a 50.0 (15)b 50.0 (15)b

4 Cancer Research 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 distrib- uted 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 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 F 2 g) and those fed 0.2% (77 F 2 g) or 0.4% (78 F 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. 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 Figure 2. Effect of feeding either 0.02% or 0.03% (w/w) 2-DG on rate of growth statistically only during the final week of the study. (A), the incidence of palpable mammary tumors that were histopathologically confirmed to be mammary carcinomas (B), and the average number of Circulating Factors histopathologically confirmed palpable mammary cancers per rat as a function of As shown in Table 3, compared with control rats, the 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 concentration of insulin (P < 0.001) in serum was significantly 7% decrease in final weight that was statistically significant (P < 0.05). Treatment reduced by 0.03% 2-DG, but not 0.02% 2-DG. Serum corticoste- with 2-DG significantly delayed the appearance of palpable mammary rone was elevated by both concentrations of 2-DG (P < 0.001). carcinomas (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 evaluated by factorial ANOVA of the absorbance data resulting from each concentration of the binding protein with increasing concentra- assay although the data are shown normalized to the control to facilitate tion of 2-DG approached statistical significance (P = 0.06). graphical presentation and visual interpretation (27). Data derived from Western ligand blot and Western blot analyses represent semiquantitative Effects of 2-Deoxyglucose on Cell Growth and ATP estimates of the amount of a specific protein that is present in serum or a Level cell extract. This fact was taken into account in the statistical evaluation of Table 4 and Fig. 3A and B show the effects of increasing the data. The data displayed in the graphs are reported as means of the ratio concentrations of 2-DG on growth of cultured MCF-7 human (experimental/control) of the actual scanning units derived from the breast cancer cells. Cell growth was significantly inhibited by Cancer Res 2005; 65: (15). August 1, 2005 7026 www.aacrjournals.org

5 2-Deoxyglucose and Mammary Carcinogenesis Table 3. Effect of 2-DG on circulating concentrations of growth factors and hormones Control 0.02% 2-DG 0.03% 2-DG Overall P Serum insulin (ng/mL) 1.43 F 0.07a 1.27 F 0.08a 0.76 F 0.09b

6 Cancer Research As shown in Fig. 2B and C and summarized in Table 2, the considered it important to investigate the cellular and molecular incidence and multiplicity of mammary carcinomas was signifi- effects of 2-DG in a highly controlled model system that is relevant cantly reduced by both 0.02% and 0.03% 2-DG and cancer latency to breast cancer. For this work, we chose the widely studied MCF-7 was prolonged. The magnitude of these effects is similar to that human breast cancer cell line. The first goal of this work was to observed in response 20% DER in the same model system (21); determine (a) if 2-DG could inhibit cell number accumulation, however, in the case of the 0.02% dose of 2-DG, the inhibition of an effect predicted by its in vivo inhibition of carcinogenesis and carcinogenesis was observed in the absence of effects of body (b) whether cellular energy charge would be reduced. As shown in weight, blood glucose, or changes in insulin, IGF-I, IGFBP-3, or Table 4, 2-DG induced a dose- and time-dependent reduction leptin and only a 20% elevation in plasma corticosterone. The in cell growth, and the data in Fig. 4 show that ATP levels were incidence of premalignant pathologies (intraductal proliferation reduced by 2-DG in a dose-dependent (P < 0.05) and time- and ductal carcinoma in situ) was also significantly reduced by both dependent (P < 0.05) manner that paralleled its effects on cell doses of 2-DG and the average number of these pathologies per rat growth. The effect on cell growth is consistent with 2-DG exerting was also reduced, but that reduction was not statistically significant. a specific effect on cell proliferation and/or apoptosis, which Collectively, these data suggest that perturbing intracellular are involved in the loss of regulation of tissue size homeostasis metabolism of glucose acts at an early stage in the disease process that results in tumor occurrence (33). The effects of 2-DG on as would be predicted based on evidence that energy production these cellular processes are currently under investigation. in transformed cells switches primarily to glycolysis, the Warberg The second goal of the in vitro work was to test three effect, and that this switch is induced by oncogene activation and/ straightforward predictions. First, based on evidence reviewed in or loss of tumor suppressor gene function (12, 13, 32). ref. (34), we predicted that the reduction in cellular energy charge Whereas the evidence presented above is supportive of our induced by 2-DG would result in the activation of AMPK. As working hypothesis that limitation of glucose availability can shown in Fig. 4, levels of phospho-AMPK were clearly elevated in inhibit the development of cancer and that this mechanism may a dose- and time-dependent manner. This finding has two account, in part, for the cancer preventive activity of DER, we implications for future in vivo investigations. The work in this 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). Cancer Res 2005; 65: (15). August 1, 2005 7028 www.aacrjournals.org

7 2-Deoxyglucose and Mammary Carcinogenesis Figure 4. Effect of 2-DG exposure on the expression of the following proteins: phosphorylated Akt, Akt, phosphorylated AMPK, AMPK, and Sirt-1. h-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 h-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). study was based on the hypothesis that DER induces a local elevated expression of Sirt-1 is a signature for DER, then we limitation in glucose availability resulting in a reduced cellular predict that if DER acts by limiting glucose availability, Sirt-1 energy charge that preferentially affects transformed cells because would be induced by treatment with 2-DG. As shown in Fig. 4, of their differential utilization of glucose relative to their Sirt-1 was induced in a dose- and time-dependent manner by nontransformed counterparts. Based on this, we predicted that 2-DG. Whether the regulation of Sirt-1 is linked to pathways of 2-DG would preferentially accumulate in these cells and inhibit which AMPK and/or Akt are components is currently under their growth. However, to test this hypothesis in vivo at the level investigation. of intracellular glucose or ATP concentrations in only trans- formed cells is difficult from a technical perspective, although the Conclusion work of Sauer and Dauchy (35) may be helpful in this regard. On A primary goal of the experiments reported in this study was to the other hand, testing the hypothesis that either DER or 2-DG determine whether a competitive inhibitor of glucose metabolism alters cellular energy charge in developing premalignant and would mimic the effects of DER by inhibiting the process of malignant cells using a surrogate marker, such as AMPK experimentally induced mammary carcinogenesis. It was antici- activation, is clearly achievable and the present work provides a pated that these experiments would also inform our working rationale for pursuing those studies. hypothesis that DER inhibits tumor development by limiting The second prediction tested in this cell culture work was that glucose availability. The in vivo and in vitro data obtained reducing cellular energy charge using 2-DG would emulate the indicate that under very restrictive conditions, 2-DG does inhibit effects of DER on cell signaling as reported in ref. (19), and that carcinogenesis without inducing many of the systemic effects that the concentration of phospho-Akt would be reduced. The data accompany DER. The in vitro data show that 2-DG reduces shown in Fig. 4 are consistent with this prediction and provide a cellular energy charge and identified three signaling pathways rationale for examining the effects of 2-DG on the signaling that seem to be modulated by limiting energy availability. These pathway of which Akt is a component in vivo under conditions in studies provide a basis for further in vivo studies of the which circulating levels of IGF-I and IGFBP-3 are not affected. mechanisms of cancer inhibition by DER and for in vitro and This is critical because it currently is thought that DER is most in vivo experiments that seek to identify ERMA that inhibit the likely to affect the activity of Akt via its effects on IGF-I signaling carcinogenic process. 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 Acknowledgments all effects are mediated through IGF-IR. Received 2/9/2005; revised 4/6/2005; accepted 5/18/2005. Finally, recently reported work has implicated the sertuin family 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 of genes, and in particular Sirt-1, as being induced by DER and in charges. This article must therefore be hereby marked advertisement in accordance accounting for some of the health benefits of DER (17, 18). If with 18 U.S.C. Section 1734 solely to indicate this fact. www.aacrjournals.org 7029 Cancer Res 2005; 65: (15). August 1, 2005

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