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2-Deoxyglucose Induces Cell Cycle Arrest and Apoptosisin Colorectal Cancer Cells Independent of Its Glycolysis Inhibition a
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Pratik Muley , Alex Olinger & Hemachand Tummala
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Department of Pharmaceutical Sciences, South Dakota State University, Brookings, South Dakota, USA Published online: 09 Mar 2015.
Click for updates To cite this article: Pratik Muley, Alex Olinger & Hemachand Tummala (2015): 2-Deoxyglucose Induces Cell Cycle Arrest and Apoptosisin Colorectal Cancer Cells Independent of Its Glycolysis Inhibition, Nutrition and Cancer, DOI: 10.1080/01635581.2015.1002626 To link to this article: http://dx.doi.org/10.1080/01635581.2015.1002626
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Nutrition and Cancer, 0(0), 1–9 Copyright Ó 2015, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2015.1002626
2-Deoxyglucose Induces Cell Cycle Arrest and Apoptosis in Colorectal Cancer Cells Independent of Its Glycolysis Inhibition Pratik Muley, Alex Olinger, and Hemachand Tummala
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Department of Pharmaceutical Sciences, South Dakota State University, Brookings, South Dakota, USA
2-Deoxyglucose (2DG) is an anticancer drug with excellent safety profile. Because of its higher dose requirements, its potential is yet to translate into a monotherapy. However, recently, 2DG has been tested as an adjunct in established chemotherapeutic regimens. 2DG enhanced the potency of several chemotherapeutic agents but not all. The rationale selection of known chemotherapeutic agents to use with 2DG is hampered becaue of the lack of complete understanding of mechanism behind 2DG anticancer effects. Although, 2DG is a well-known glycolytic inhibitor, which inhibits the key glycolytic enzyme hexokinase, its anticancer effects cannot be fully explained by this simplistic mechanism alone. In this article, we have shown for the first time that 2DG induced a transient expression of p21 and a continuous expression of p53 in colorectal cancer cells (SW620). The treatment also caused cell cycle arrest at G0/G1 phase and induced apoptosis through the mitochondrial pathway. The effects of 2DG on p21 and p53 protein levels were totally independent of its inhibitory effect on either hexokinase or ATP levels. Results from this study provides key insights into novel molecular mechanisms of 2DG and directs rational selection of other anticancer drugs to combine with 2DG in colorectal cancer treatment.
INTRODUCTION One of the earliest observations regarding the biochemical differences between the metabolism of cancer and normal cell was made by Warburg in 1930 (1). He observed that there is a metabolic shift from oxidative phosphorylation to aerobic glycolysis to generate ATP as an energy source in the majority of the cancer cells and termed as metabolic remodeling or adaptation (reviewed in Ref. 2). This unique metabolic predisposition is the most fundamental metabolic alteration in malignant Submitted 20 January 2014; accepted in final form 17 December 2014. Address correspondence to Hemachand Tummala, Department of Pharmaceutical Sciences, South Dakota State University, 1 Administration Lane, Brookings, SD 57007. E-mail: hemachand.tummala@ sdstate.edu Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/hnuc.
vs. normal cells and therefore, garnered a considerable attention as a strategy to target drugs against cancer (3). Accordingly, various small molecule inhibitors of glycolytic pathway have been used effectively in the past to halt the progression of cancer both in-vitro and in-vivo (4–6). 2-Deoxy glucose (2DG) is one of the well-characterized small molecular inhibitor of glycolysis both in animals and humans (7,8). It is very safe and well tolerated (the oral LD50 of 2DG is >8000 mg/ kg in mice and rats) (7). 2DG is a derivative of glucose that lacks hydroxyl group at the second carbon atom. During glycolysis, 2DG is converted into 2DG-6-phosphate by the enzyme hexokinase. However, because of the lack of hydroxyl group at second carbon, it cannot be further metabolized and therefore accumulates inside the cell and inhibits the rate limiting glycolytic enzyme, hexokinase, in the process (9). The direct cellular consequences of 2DG treatment are the depletion of intracellular ATP through the inhibition of glycolysis, and thereby, suppression of cell growth (10,11). However, several recent observations question this simplistic assumption. Firstly, the decline in ATP levels following 2DG treatment in eukaryotes is moderate at best (12). Secondly, tumor cells react differently to 2DG than its fluorescent analogue 2-fluorodeoxy-D-glucose (FDG). Although FDG is a stronger inhibitor of glycolysis, it is nontoxic to cells that are responsive to 2DG (13). Taken together, these observations warrant the existence of additional cellular effects or targets for 2DG beyond its glycolytic inhibition. The success of 2DG as a single chemotherapeutic agent was limited by its high-dose requirements (14,15). However, 2DG has been previously used as a dietary supplement in various studies (16,17). Because of its excellent safety profile, 2DG has potential to be used as a supplemental therapy to enhance the efficiency of already existing chemotherapeutic agents. Therefore, understanding the additional mechanisms of 2DG would enable scientists to design new combination therapies for cancer with 2DG and other anticancer drugs with similar molecular mechanisms. This will increase the efficacy of traditional anticancer drugs thereby decreasing their dose and its 1
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associated toxicity. In this work, we have evaluated the additional cellular effects of 2DG, apart from inhibiting glycolysis, in colon cancer cell lines. Our findings indicate that 2DG increases the expression of p21 and p53 in colorectal cancer cell lines leading to cell cycle arrest at the G0/G1 phase. This increase is independent of glycolytic inhibition caused by 2DG. The inhibition of cell cycle was followed by apoptotic cell death via intrinsic pathway involving increased expression of Bax and mitochondrial depolarization.
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MATERIALS AND METHODS Reagents and Chemicals All the cell lines used in the article were purchased from ATCC (Manassas, VA). All the cell culture media (RPMI 1640, DMEM, and Leibovitz’s L-15), fetal calf serum (FCS), penicillin, streptomycin, and trypsin were purchased from Fisher Scientific (Waltham, MA). 2DG, MTT (3,4,5-dimethiazol-2,5-diphenyl-tetrazolium bromide), JC1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolocarbocyanine iodide) and all other chemicals were purchased from Sigma (St. Louis, MO). Antibodies against proteins p21, cyclin dependent kinase [CDKs (CDK4, and CDK2)], Bax, cytochrome c, cleaved PARP, cyclins D1, A, and E were purchased from SantaCruz Biotechnologies (Santa Cruz, CA). Antibody against betatubulin was purchased from the Developmental Hybridoma Bank (Iowa City, IA). Chemiluminescence detection kit was purchased from Amersham (Pittsburgh, PA). The TdT-mediated dUTP-biotin nick end labeling (TUNEL) kit was purchased from Millipore (Billerica, MA).
Cell Culture and 2DG Treatment Three human malignant colorectal cancer cell lines (Gc3/ C1, SW480, and SW620), 2 human breast cancer cell lines (MDA-MB-231 and MCF7), human ovarian cancer cell line (Ovcar 3), mouse melanoma cell line (B16F10), and human kidney epithelial cell line (HEK 293) were used in this study. The cells were grown in RPMI 1640 (Ovcar 3 and Gc3/C1), DMEM (MCF 7, MDA-MB-231, B16F10, and HEK293) or Leibovitz’s L-15 medium (SW620 and SW480) supplemented with 10% FCS, penicillin (100U/ml), and streptomycin (100 mg/ml) in a humidified incubator (370C, 5% CO2). A 1 M stock solution of 2DG was prepared in 50 mM phosphate buffered saline (PBS), stored at ¡20 C and diluted in the complete media to obtain the desired concentrations.
Cytoxicity Assays and the Determination of IC50 The dose of 2DG required to kill 50% of cells (IC50) in various cell lines was determined by MTT (3,4,5-dimethiazol-2, 5-diphenyl-tetrazolium bromide) assay. Ten thousand cells were seeded per well of a 96-well plate and allowed to adhere
for 24 h. After incubating the cells for 72 h with various concentrations of 2DG (0–25 mM), the viability of the cells was assessed using MTT assay. The IC50 was calculated using SigmaPlotÒ software by plotting the cell viability against the concentration of 2DG. All the experiments were performed in replicates of 3 with 3 repetitions.
Protein Isolation and Western Blotting Cells were treated with the various concentrations of 2DG in a 6-well plate and incubated for different time periods following which, total cell protein was extracted using 0.25 mL of cold fresh lysis buffer [1% Triton X-100, 150 mM NaCl, 0.5 mM MgCl2, 200 mM EGTA, and 50 mM Tris-HCl (pH 7.4) with aprotinin (2 mg/mL), DTT (2 mM), and phenylmethylsulfonyl fluoride (PMSF, 1 mM)]. Proteins from the cell lysates were separated on a 10–12% polyacrylamide gel under reducing conditions (reducing-SDS-PAGE). Proteins were then transferred onto a nitrocellulose membrane, and the blot was blocked with 5% nonfat milk. The protein levels were detected by immunodetection with specific antibodies. Antibodies against proteins, p21, CDK4, CDK2, Bax, Cytochrome c, cleaved PARP, and tubulin were used at a concentration of 1:1000 dilution. Anticyclin D1, A, and E polyclonal antibodies were used at a concentration of 1:250 dilution. Appropriate HRP-conjugated secondary antibodies were incubated at room temperature at a dilution of 1:5000. The specific protein complexes were identified using chemiluminescence detection kit.
Preparation of Mitochondrial and Cytosolic Fractions (18,19) The cells were trypsinized and washed with cold PBS. The cell pellet was resuspended in ice cold isolation buffer (0.3 M mannitol, 0.1% BSA, 0.2 mM EDTA, 10 mM HEPES, pH 7.4). The cells were then homogenized using a 2-ml glass homoginizer (Dounce: loosen 5 times then tighten 5 times). The cell homogenate was centrifuged at 1000g at 4 C for 10 min. The supernatant was centrifuged at 14,000g for 15 min at 4 C, and the resulting supernatant was saved as the cytosolic fraction. The pellet containing the mitochondria was further washed twice with ice cold isolation buffer. Protein content in both cytosolic and mitochondrial extracts was determined using bicinchoninic acid assay kit following a standard protocol.
Cell Cycle Analysis The cell cycle analysis was performed following the method described in the literature (20). In brief, SW620 cells were switched to a media without serum for a period of 24 h, to render them quiescent and synchronize their cell cycle phases. Following this, the cells were treated with various concentrations of 2DG for 18 h and harvested by trypsinization.
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The harvested cells were washed twice with PBS and fixed in 70% ethanol at 4 C. The nuclear DNA was stained using propidium iodide (50 Fg/ml) in the presence of DNase free RNase (2 U/mL). The cells were sorted depending on the DNA content using a fluorescence activated cell sorter machine (BectonDickinson, San Jose, CA). The proportion of nuclei in each phase of the cell cycle was determined using CellFIT software (BectonDickinson, San Jose, CA). TUNEL Staining TdT-mediated dUTP-biotin nick end labeling (TUNEL) kit was used to detect apoptotic cells that had undergone caspasedependant genomic fragmentation. Briefly, SW620 cells were seeded in 35-mm culture plates and treated with various concentrations of 2DG and appropriate controls for 72 h. Following which, the cells were harvested, fixed, and stained by following the manufacturer’s protocol. The fraction of TUNEL positive cells was determined using flowcytometric analysis. Mitochondrial Potential The mitochondrial potential of the cells after treatment with various doses of 2DG was determined using JC1 (5,50 , 6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolocarbocyanine iodide) staining. The cells were incubated with 2DG for 18 h following which the mitochondria were stained with JC1 following the manufacturers protocol. The ratio of red:green fluorescence was determined using the excitation emission wavelengths of 525 nm and 590 nm for red fluorescence and 490 nm and 530 nm for green fluorescence, respectively. Image and Statistical Analysis The Western blot band quantification was done using ImageJ software. Student’s t-test (paired 2-tailed) was used to determine the statistical significance. The data was considered significant if the P value was less than 0.05. RESULTS Colorectal Cancer Cells Are More Susceptible to 2DG Treatment The cytotoxic effect of 2DG on various malignant and immortalized cell lines was tested using MTT assay. The types of cell lines tested were immortalized kidney epithelial cell line (HEK293), colorectal cancer cell lines (SW620, SW480, and GC3/Cl), breast cancer cell lines (MDA-MB-231 and MCF7), ovarian cancer cell line (Ovcar 3), and mouse melanoma cell line (B16F10). The IC50 of 2DG on various cell lines is represented in Fig. 1. Out of eight cell lines tested, 2DG exerts the least cytotoxic effect on cell line originated from noncancerous immortalized cells (HEK 293, IC50:
FIG. 1. The sensitivity of various cell lines to 2-deoxyglucose (2DG) treatment. The IC50 of 2DG was calculated in various cell lines after treating with 2DG for 72 h. The colorectal cancer cell lines SW620, SW480, and GC3/Cl exhibit a lower IC50 value compared to other cell lines. The data represents the mean § SD of 3 replicates.
6.452 mM), which further supports its more selectivity toward cancer cell lines. The IC50 values of the 3 colorectal cancer cell lines were lower than that of other cancer cell lines tested. The metastatic breast cancer cells (MDA-MB-231, IC50 D 1.874 mM) are more susceptible to 2DG treatment than nonmetastatic breast cancer cells (MCF7a, IC50 D 5.891 mM). Taken together, it is clear that colorectal cancer cells are more susceptible to 2DG treatment than other cancer cells tested. Therefore, we have continued to use colorectal cancer cells (SW620) for further analysis of 2DG activity. 2DG inhibits the progression of cell cycle in colorectal cancer cell lines. The reduced viability of cancer cells after 2DG treatment could be interpreted as a result of inhibiting cell cycle progression or inducing cell death or both. Therefore, the role of 2DG in modulating the cell cycle progression was evaluated in SW620 cell line using flow cytometric analysis as described in Methods. Treatment of SW620 cells with 2DG for 18 h caused cell cycle arrest at the G0/G1 phase (Fig. 2A and 2B). The percent of cells in G0/G1 phase have significantly increased from 62.49% to 80.15% (P < 0.05; student ttest) after 2DG treatment. Simultaneously the proportion of cells in S phase has decreased from 19.11% to 7.13%.
2DG Alters the Expression of Various Cell Cycle Associated Proteins Expression levels of various proteins involved in cell cycle are critical for proper progression of cell cycle (17). Treatment with 2DG decreased the expression of various proteins associated with cell cycle progression (Fig. 3). 2DG treatment caused a downregulation of cyclin A, cyclin D1, and CDK4, whereas the levels of CDK2 and cyclin E remained unaltered.
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FIG. 3. Relative expression of various cell cycle proteins and relative density measurements. SW620 cells were treated with various doses of 2-deoxyglucose (2DG) for 18 h. The expression levels of various cell cycle associated proteins were checked by Western blot analysis followed by measuring pixel density using ImageJ software (n D 3). The figure is a representative image of 3 separate experiments. (P < 0.05; n D 3).
FIG. 2. Cell cycle analysis of SW620 cells after 2-deoxyglucose (2DG) treatment. Control SW620 cells (A) or after treatment with 20 mM 2DG for 18 h (B) were fixed and stained with PI and analyzed for DNA content using flow cytometer. The data represents mean percent of cells in a particular cell cycle phase § SD (n D 3).
Such a reduced expression of CDK4 and other cell cycle associate proteins may be responsible, in part, for the G0/G1 cell cycle arrest observed after 2DG treatment (Fig. 2).
2DG Increases the Expression of p21 and p53 Proteins The protein p21 is one of the key CDK-inhibitory proteins exerting a regulatory role in cell cycle progression. The expression of p21 can be p53-mediated or p53-independent. Treatment with 2DG increased the expression of p21 as well as p53 in SW620 cell lines analyzed by Western blot analysis (Fig. 4A). This magnitude of increase was dependent on the dose of 2DG (Fig. 4A). The kinetics of p21 and p53
expression in 2DG treated (5 mM) SW620 cells was analyzed by Western blot analysis. 2DG dependent increased expression of p21 was transient with the maximum expression occurring after 18 h of treatment (Fig. 4B). However, the protein levels of p53 increased even after 18 h of 2DG treatment and remained same for at least 48 h.
2DG Causes Apoptosis in SW620 Cells Through Intrinsic Pathway Cell cycle arrest at G0/G1 phase and the transient over expression of p21 protein may direct the cells towards apoptosis (21,22). To check whether the treatment of 2DG causes the induction of apoptosis, SW620 cells were treated with various doses of 2DG and the proportion of apoptotic cells was analyzed using TUNEL assay. 2DG treatment in SW620 cells caused an increase in TUNEL positive cells. Around 18% and 50% of cells showed TUNEL staining after treating with 10 mM and 20 mM 2DG, respectively (Fig. 5A–C). The involvement of apoptosis was further confirmed by increased cleavage of PARP after 2DG treatment (Fig. 5D). Apoptosis could occur either by intrinsic or extrinsic pathways. The
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FIG. 4. Expression of p21 and p53 after 2-deoxyglucose (2DG) treatment in SW620 cells. A: The SW620 cells were treated with various concentrations of 2DG for 48 h and amounts of p21 and p53 proteins expressed in the cell lysates were analyzed by Western blot analysis. B: Cells were treated with 5 mM of 2DG for indicated times and the amount of p21 and p53 proteins expressed were analyzed by Western blot analysis. The relative density of various protein levels normalized to b-Tubulin were plotted. The figure is a representative image of 3 separate experiments (* and #P < 0.05; n D 3).
following sets of results indicate that 2DG caused apoptosis in colon cancer cells (SW620) through intrinsic or mitochondrial pathway. Treating the SW620 cells with 2DG caused 1) increased expression of pro-apoptotic protein BAX (Fig. 6A), 2) release of cytochrome-C from the mitochondria into cytosol (Fig. 6B), and 3) loss of mitochondrial potential (Fig. 6C). The enrichment of subcellular fractions was confirmed by Western blot analysis with organelle specific marker proteins (Fig. 6D).
2DG Dependent Overexpression of p21 and p53 Is Independent of Glycolytic Inhibition 2DG is known to inhibit glycolysis. Therefore, the effect of 2DG on expression levels of p21 and p53 could be an indirect effect of 2DG due its inhibition on glycolysis or through an independent unknown mechanism. To address this, another glycolysis inhibitor, 3-bromopyruvate (3BP) was used to inhibit glycolysis (20). Similar to 2DG, 3BP is also a wellknown inhibitor of an enzyme hexokinase that is involved in glycolysis. Inhibition of glycolysis by 3BP did not increase the expression of p21 and p53 proteins (Fig. 7). This data clearly indicates that inhibiting the hexokinase enzyme by 2DG and thereby glycolysis in colorectal cancer cells may not be responsible for its effects on the protein levels of p21 or p53.
DISCUSSION The biggest drawback of current cancer chemotherapy is the extensive tissue toxicity of conventional chemotherapeutic agents (21). Therefore, preferential killing of cancer cells without significant toxicity to normal cells is an important consideration for chemotherapy. Cancer cells are metabolically more active and depend on glycolysis as an energy source for their survival. 2DG is a small molecular inhibitor of glycolysis with very good safety index (23–25). Recent studies have indicated that the glycolytic inhibition effect of 2DG alone will not explain its success in various preclinical and clinical studies in cancer (26,27). In this article, we have investigated additional mechanisms by which 2DG mediates its cytotoxic or antiproliferative actions in colorectal cancer cells. 2DG accumulates predominantly in tumors but does not harm other tissues. Despite its safety profile and preference to cancer, its success is yet to be translated to clinical application due to its high dose requirement as a single agent (65–100 mg/ Kg body weight) (8,11). However, recently, 2-DG has been tried as an effective adjuvant for chemotherapeutic agents to enhance their efficacy (28,29). Although, 2DG enhanced the efficacy of several chemotherapeutic agents, it has also lowered the effectiveness of other treatments (30). Therefore, the success of the combination therapy with 2DG may depend on the selection of the appropriate chemotherapeutic agent. We hypothesize that the understanding of molecular mechanisms
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FIG. 5. Detection of apoptosis after 2-deoxyglucose (2DG) treatment. SW620 cells were treated with PBS (A), 10 mM of 2DG (B), or 20 mM of 2DG (C) for 72 h (n D 3). The proportion of apoptotic cells were identified by TUNEL staining followed by flowcytometric analysis. D: Expression of cleaved PARP after treatment with various concentrations of 2DG for 72 h in SW620 cells. The relative density of various protein levels normalized to b-Tubulin were plotted. The figure is a representative image of 3 separate experiments (*P < 0.05; n D 3).
behind 2DG effectiveness will help in judicial selection of the combination drug for cancer treatment. In this article, we have shown that 2DG modulates the expression of various proteins involved in the regulation of cell cycle (Fig. 3). The expression of p21 protein is enhanced transiently by 2DG. In contrast, enhanced expression of p53 was continued for at least 48 h. By modulating the expression of proteins that regulate cell cycle (Fig. 3), 2DG arrested the cell cycle in colon cancer cells at G0/G1 phase. CDKs plays a critical role in the progression of cell cycle beyond G1 check point, especially CDK2 and CDK4, and are active targets for anticancer drugs (31). Although, the cell cycle was arrested at the G0/G1 phase, the level of CDK2 remained unaltered. In contrast, the protein levels of CDK4 were significantly reduced. This observation is consistent with previous reports on colorectal cancer cell lines that activity of CDK2 is dispensable for cell cycle progression and inhibition of CDK4 is sufficient to cause G0/G1 cell cycle arrest (32,33). Thus, the cell cycle arrest at the G1 phase caused by the 2DG treatment is consistent with the decreased CDK4 and Cyclin D1 levels.
In addition to cell cycle arrest, 2DG has also induced apoptosis in colon cancer cells. Apoptosis typically proceeds by either the intrinsic pathway through the release of cytochrome c from the mitochondria into cytosol to activate apoptosomes or the extrinsic pathway, in which caspases are activated directly downstream of death receptors (34,35). In colon cancer cells, 2DG induced apoptosis through intrinsic pathway involving mitochondria. This finding is consistent with previous observations that 2DG has successfully been used to sensitize cancer cells to agents that alter mitochondrial function such as mitochondrial inhibitors (oligomycin and antimycin) (36), delocalized cationic compounds (mito-Q) (36), and Bcl2 antagonists (ABT 263 and ABT 737) (36). Importantly, some of these sensitizing effects of 2DG have been shown to be independent of glucose depletion. These observations indicate that the anticancer effects of 2DG can be attributed at least in part to mechanisms other than interfering with glycolysis through hexokinase inhibition. Thus, the knowledge of the additional mechanisms of 2DG on cell cycle and mitochondrial driven apoptosis will provide
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FIG. 6. Levels of Bax, Cytochrome c, and mitochondrial potential after 2-deoxyglucose (2DG) treatment. SW620 cells were treated with different doses of 2DG and the levels of Bax (A) and Cytochrome c (B) expression were analyzed using Western blot analysis. C: The mitochondrial potential was determined using JC1 dye after 2DG treatment by analyzing the red:green fluorescence in SW620 cells. D: Cox IV and a-tubulin, which are markers for mitochondria and cytosol, respectively, were used to show the enrichment of mitochondrial and cytosolic fractions.
crucial information for selecting suitable chemotherapeutic combinations to maximize the therapeutic efficacy. 2DG is known to inhibit glycolysis. The novel pathways that are affected by 2DG (p21, p53 expression) as observed in this article, could be indirect effects of its inhibition of glycolysis or completely independent molecular events. Cancer cells predominantly depend on ATP generated by glycolysis for their energy source. 2DG inhibits hexokinase, a key enzyme in glycolysis and, thus, starves cancer cells of ATP. The effect of 2DG observed in this study could be a direct result of either ATP starvation or hexokinase inhibition. The mechanisms are dissected by physiological and pharmacological approaches (Fig. 7). To simulate the ATP starvation in cancer cells without inhibiting glycolysis, colon cancer cells were grown in media in which glucose was replaced by galactose. In the presence of galactose, the cells are unable to generate ATP using glycolysis. During glycolysis, 1 glucose molecule is converted to pyruvate and generates 2 molecules of ATP, whereas galactose enters glycolysis through galactose phosphate and generates no net ATP. Therefore, in presence of galactose, the glycolytic pathway is not inhibited, but the
production of ATP is halted for cancer cells (37). We observed that replacement of glucose in the media by galactose did not induce a significant cell death (data not shown) and also had no effect on the levels of p21 or p53 (data not shown). This observation clearly indicates that the effects of 2DG on colon cancer cells are not due to mere glycolytic ATP starvation. However, the effects could be a result of direct hexokinase inhibition by 2DG. To simulate the inhibition of glycolysis through hexokinase inhibition, the cells were treated with 3-bromopyruvate, another potent inhibitor of hexokinase. The purpose of the experiment is not to compare the efficiencies of 3BP and 2DG as anticancer drugs, but to use 3BP as a biochemical tool to inhibit hexokinase. Although both 2DG and 3BP inhibit hexokinase, only 2DG altered the levels of p21 and p53 in colorectal cell line. Taken together, the experiments with galactose and 3BP strongly suggest that the effects of 2DG on p21/p53 pathway are independent molecular events beyond ATP starvation or hexokinase inhibition. Understanding these additional mechanisms will most certainly enable us to judicially choose right combination of anti-cancer drugs to use with 2DG.
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FIG. 7. Protein levels of p21 and p53 after 3-Bromopyruvate treatment. SW620 cells were treated with different doses of 3BP and the levels of p21 and p53 expression were analyzed using Western blot analysis.
CONCLUSIONS The work proposes an additional molecular mechanism of anticancer activity of 2DG in colorectal cancer cell lines. The findings of the work also open up possibilities of novel combination therapies of agents affecting the p21/p53 protein system along with 2DG in colorectal cancer such as doxorubicin (38). Also, with the current knowledge in formulation design, these novel drug therapies could be targeted to the colon to exert maximum efficacy and reduce the overall untoward effects.
ACKNOWLEDGEMENTS We are thankful to Dr. Bhimanna Kuppast for his valuable suggestions and expert comments on the work.
FUNDING We also thank the Department of Pharmaceutical Sciences, South Dakota State University for funding the study.
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2DG INDUCES G0/G1 ARREST INDEPENDENT OF GLYCOLYTIC BLOCK 21. Vermeulen K, Van Bockstaele DR, and Berneman ZN: The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif 36, 131–149, 2003. 22. Gartel AL and Tyner AL: The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther 1(8), 639–649, 2002. 23. Han Z, Wei W, Dunaway S, Darnowski JW, Calabresi P, et al.: Role of p21 in apoptosis and senescence of human colon cancer cells treated with camptothecin. J Biol Chem 277, 17154–17160, 2002. 24. Ihrlund LS, Hernlund E, Khan O, and Shoshan MC: 3-Bromopyruvate as inhibitor of tumour cell energy metabolism and chemopotentiator of platinum drugs. Mol Oncol 2, 94–101, 2008. 25. Duffy MJ: The war on cancer: are we winning? Tumour Biol 34, 1275–1284, 2013. 26. Reutter M, Emons G, and Gr€undker C: Starving tumors: inhibition of glycolysis reduces viability of human endometrial and ovarian cancer cells and enhances antitumor efficacy of GnRH receptor-targeted therapies. Int J Gynecol Cancer 23, 34–40, 2013. 27. Sottnik JL, Lori JC, Rose BJ, and Thamm DH: Glycolysis inhibition by 2deoxy-D-glucose reverts the metastatic phenotype in vitro and in vivo. Clin Exp Metastasis 28, 865–875, 2011. 28. Cheong JH, Park ES, Liang J, Dennison JB, Tsavachidou D, et al.: Dual inhibition of tumor energy pathway by 2-deoxyglucose and metformin is effective against a broad spectrum of preclinical cancer models. Mol Cancer Ther 10, 2350–2362, 2011. 29. Zhong D, Liu X, Schafer-Hales K, Marcus AI, Khuri FR, et al.: 2-Deoxyglucose induces Akt phosphorylation via a mechanism independent of LKB1/AMP-activated protein kinase signaling activation or glycolysis inhibition. Mol Cancer Ther 7, 809–917, 2008.
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2-Deoxyglucose Induces Cell Cycle Arrest and Apoptosisin Colorectal Cancer Cells Independent of Its Glycolysis Inhibition a
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Pratik Muley , Alex Olinger & Hemachand Tummala
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Department of Pharmaceutical Sciences, South Dakota State University, Brookings, South Dakota, USA Published online: 09 Mar 2015.
Click for updates To cite this article: Pratik Muley, Alex Olinger & Hemachand Tummala (2015): 2-Deoxyglucose Induces Cell Cycle Arrest and Apoptosisin Colorectal Cancer Cells Independent of Its Glycolysis Inhibition, Nutrition and Cancer, DOI: 10.1080/01635581.2015.1002626 To link to this article: http://dx.doi.org/10.1080/01635581.2015.1002626
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Nutrition and Cancer, 0(0), 1–9 Copyright Ó 2015, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2015.1002626
2-Deoxyglucose Induces Cell Cycle Arrest and Apoptosis in Colorectal Cancer Cells Independent of Its Glycolysis Inhibition Pratik Muley, Alex Olinger, and Hemachand Tummala
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Department of Pharmaceutical Sciences, South Dakota State University, Brookings, South Dakota, USA
2-Deoxyglucose (2DG) is an anticancer drug with excellent safety profile. Because of its higher dose requirements, its potential is yet to translate into a monotherapy. However, recently, 2DG has been tested as an adjunct in established chemotherapeutic regimens. 2DG enhanced the potency of several chemotherapeutic agents but not all. The rationale selection of known chemotherapeutic agents to use with 2DG is hampered becaue of the lack of complete understanding of mechanism behind 2DG anticancer effects. Although, 2DG is a well-known glycolytic inhibitor, which inhibits the key glycolytic enzyme hexokinase, its anticancer effects cannot be fully explained by this simplistic mechanism alone. In this article, we have shown for the first time that 2DG induced a transient expression of p21 and a continuous expression of p53 in colorectal cancer cells (SW620). The treatment also caused cell cycle arrest at G0/G1 phase and induced apoptosis through the mitochondrial pathway. The effects of 2DG on p21 and p53 protein levels were totally independent of its inhibitory effect on either hexokinase or ATP levels. Results from this study provides key insights into novel molecular mechanisms of 2DG and directs rational selection of other anticancer drugs to combine with 2DG in colorectal cancer treatment.
INTRODUCTION One of the earliest observations regarding the biochemical differences between the metabolism of cancer and normal cell was made by Warburg in 1930 (1). He observed that there is a metabolic shift from oxidative phosphorylation to aerobic glycolysis to generate ATP as an energy source in the majority of the cancer cells and termed as metabolic remodeling or adaptation (reviewed in Ref. 2). This unique metabolic predisposition is the most fundamental metabolic alteration in malignant Submitted 20 January 2014; accepted in final form 17 December 2014. Address correspondence to Hemachand Tummala, Department of Pharmaceutical Sciences, South Dakota State University, 1 Administration Lane, Brookings, SD 57007. E-mail: hemachand.tummala@ sdstate.edu Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/hnuc.
vs. normal cells and therefore, garnered a considerable attention as a strategy to target drugs against cancer (3). Accordingly, various small molecule inhibitors of glycolytic pathway have been used effectively in the past to halt the progression of cancer both in-vitro and in-vivo (4–6). 2-Deoxy glucose (2DG) is one of the well-characterized small molecular inhibitor of glycolysis both in animals and humans (7,8). It is very safe and well tolerated (the oral LD50 of 2DG is >8000 mg/ kg in mice and rats) (7). 2DG is a derivative of glucose that lacks hydroxyl group at the second carbon atom. During glycolysis, 2DG is converted into 2DG-6-phosphate by the enzyme hexokinase. However, because of the lack of hydroxyl group at second carbon, it cannot be further metabolized and therefore accumulates inside the cell and inhibits the rate limiting glycolytic enzyme, hexokinase, in the process (9). The direct cellular consequences of 2DG treatment are the depletion of intracellular ATP through the inhibition of glycolysis, and thereby, suppression of cell growth (10,11). However, several recent observations question this simplistic assumption. Firstly, the decline in ATP levels following 2DG treatment in eukaryotes is moderate at best (12). Secondly, tumor cells react differently to 2DG than its fluorescent analogue 2-fluorodeoxy-D-glucose (FDG). Although FDG is a stronger inhibitor of glycolysis, it is nontoxic to cells that are responsive to 2DG (13). Taken together, these observations warrant the existence of additional cellular effects or targets for 2DG beyond its glycolytic inhibition. The success of 2DG as a single chemotherapeutic agent was limited by its high-dose requirements (14,15). However, 2DG has been previously used as a dietary supplement in various studies (16,17). Because of its excellent safety profile, 2DG has potential to be used as a supplemental therapy to enhance the efficiency of already existing chemotherapeutic agents. Therefore, understanding the additional mechanisms of 2DG would enable scientists to design new combination therapies for cancer with 2DG and other anticancer drugs with similar molecular mechanisms. This will increase the efficacy of traditional anticancer drugs thereby decreasing their dose and its 1
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associated toxicity. In this work, we have evaluated the additional cellular effects of 2DG, apart from inhibiting glycolysis, in colon cancer cell lines. Our findings indicate that 2DG increases the expression of p21 and p53 in colorectal cancer cell lines leading to cell cycle arrest at the G0/G1 phase. This increase is independent of glycolytic inhibition caused by 2DG. The inhibition of cell cycle was followed by apoptotic cell death via intrinsic pathway involving increased expression of Bax and mitochondrial depolarization.
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MATERIALS AND METHODS Reagents and Chemicals All the cell lines used in the article were purchased from ATCC (Manassas, VA). All the cell culture media (RPMI 1640, DMEM, and Leibovitz’s L-15), fetal calf serum (FCS), penicillin, streptomycin, and trypsin were purchased from Fisher Scientific (Waltham, MA). 2DG, MTT (3,4,5-dimethiazol-2,5-diphenyl-tetrazolium bromide), JC1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolocarbocyanine iodide) and all other chemicals were purchased from Sigma (St. Louis, MO). Antibodies against proteins p21, cyclin dependent kinase [CDKs (CDK4, and CDK2)], Bax, cytochrome c, cleaved PARP, cyclins D1, A, and E were purchased from SantaCruz Biotechnologies (Santa Cruz, CA). Antibody against betatubulin was purchased from the Developmental Hybridoma Bank (Iowa City, IA). Chemiluminescence detection kit was purchased from Amersham (Pittsburgh, PA). The TdT-mediated dUTP-biotin nick end labeling (TUNEL) kit was purchased from Millipore (Billerica, MA).
Cell Culture and 2DG Treatment Three human malignant colorectal cancer cell lines (Gc3/ C1, SW480, and SW620), 2 human breast cancer cell lines (MDA-MB-231 and MCF7), human ovarian cancer cell line (Ovcar 3), mouse melanoma cell line (B16F10), and human kidney epithelial cell line (HEK 293) were used in this study. The cells were grown in RPMI 1640 (Ovcar 3 and Gc3/C1), DMEM (MCF 7, MDA-MB-231, B16F10, and HEK293) or Leibovitz’s L-15 medium (SW620 and SW480) supplemented with 10% FCS, penicillin (100U/ml), and streptomycin (100 mg/ml) in a humidified incubator (370C, 5% CO2). A 1 M stock solution of 2DG was prepared in 50 mM phosphate buffered saline (PBS), stored at ¡20 C and diluted in the complete media to obtain the desired concentrations.
Cytoxicity Assays and the Determination of IC50 The dose of 2DG required to kill 50% of cells (IC50) in various cell lines was determined by MTT (3,4,5-dimethiazol-2, 5-diphenyl-tetrazolium bromide) assay. Ten thousand cells were seeded per well of a 96-well plate and allowed to adhere
for 24 h. After incubating the cells for 72 h with various concentrations of 2DG (0–25 mM), the viability of the cells was assessed using MTT assay. The IC50 was calculated using SigmaPlotÒ software by plotting the cell viability against the concentration of 2DG. All the experiments were performed in replicates of 3 with 3 repetitions.
Protein Isolation and Western Blotting Cells were treated with the various concentrations of 2DG in a 6-well plate and incubated for different time periods following which, total cell protein was extracted using 0.25 mL of cold fresh lysis buffer [1% Triton X-100, 150 mM NaCl, 0.5 mM MgCl2, 200 mM EGTA, and 50 mM Tris-HCl (pH 7.4) with aprotinin (2 mg/mL), DTT (2 mM), and phenylmethylsulfonyl fluoride (PMSF, 1 mM)]. Proteins from the cell lysates were separated on a 10–12% polyacrylamide gel under reducing conditions (reducing-SDS-PAGE). Proteins were then transferred onto a nitrocellulose membrane, and the blot was blocked with 5% nonfat milk. The protein levels were detected by immunodetection with specific antibodies. Antibodies against proteins, p21, CDK4, CDK2, Bax, Cytochrome c, cleaved PARP, and tubulin were used at a concentration of 1:1000 dilution. Anticyclin D1, A, and E polyclonal antibodies were used at a concentration of 1:250 dilution. Appropriate HRP-conjugated secondary antibodies were incubated at room temperature at a dilution of 1:5000. The specific protein complexes were identified using chemiluminescence detection kit.
Preparation of Mitochondrial and Cytosolic Fractions (18,19) The cells were trypsinized and washed with cold PBS. The cell pellet was resuspended in ice cold isolation buffer (0.3 M mannitol, 0.1% BSA, 0.2 mM EDTA, 10 mM HEPES, pH 7.4). The cells were then homogenized using a 2-ml glass homoginizer (Dounce: loosen 5 times then tighten 5 times). The cell homogenate was centrifuged at 1000g at 4 C for 10 min. The supernatant was centrifuged at 14,000g for 15 min at 4 C, and the resulting supernatant was saved as the cytosolic fraction. The pellet containing the mitochondria was further washed twice with ice cold isolation buffer. Protein content in both cytosolic and mitochondrial extracts was determined using bicinchoninic acid assay kit following a standard protocol.
Cell Cycle Analysis The cell cycle analysis was performed following the method described in the literature (20). In brief, SW620 cells were switched to a media without serum for a period of 24 h, to render them quiescent and synchronize their cell cycle phases. Following this, the cells were treated with various concentrations of 2DG for 18 h and harvested by trypsinization.
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The harvested cells were washed twice with PBS and fixed in 70% ethanol at 4 C. The nuclear DNA was stained using propidium iodide (50 Fg/ml) in the presence of DNase free RNase (2 U/mL). The cells were sorted depending on the DNA content using a fluorescence activated cell sorter machine (BectonDickinson, San Jose, CA). The proportion of nuclei in each phase of the cell cycle was determined using CellFIT software (BectonDickinson, San Jose, CA). TUNEL Staining TdT-mediated dUTP-biotin nick end labeling (TUNEL) kit was used to detect apoptotic cells that had undergone caspasedependant genomic fragmentation. Briefly, SW620 cells were seeded in 35-mm culture plates and treated with various concentrations of 2DG and appropriate controls for 72 h. Following which, the cells were harvested, fixed, and stained by following the manufacturer’s protocol. The fraction of TUNEL positive cells was determined using flowcytometric analysis. Mitochondrial Potential The mitochondrial potential of the cells after treatment with various doses of 2DG was determined using JC1 (5,50 , 6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolocarbocyanine iodide) staining. The cells were incubated with 2DG for 18 h following which the mitochondria were stained with JC1 following the manufacturers protocol. The ratio of red:green fluorescence was determined using the excitation emission wavelengths of 525 nm and 590 nm for red fluorescence and 490 nm and 530 nm for green fluorescence, respectively. Image and Statistical Analysis The Western blot band quantification was done using ImageJ software. Student’s t-test (paired 2-tailed) was used to determine the statistical significance. The data was considered significant if the P value was less than 0.05. RESULTS Colorectal Cancer Cells Are More Susceptible to 2DG Treatment The cytotoxic effect of 2DG on various malignant and immortalized cell lines was tested using MTT assay. The types of cell lines tested were immortalized kidney epithelial cell line (HEK293), colorectal cancer cell lines (SW620, SW480, and GC3/Cl), breast cancer cell lines (MDA-MB-231 and MCF7), ovarian cancer cell line (Ovcar 3), and mouse melanoma cell line (B16F10). The IC50 of 2DG on various cell lines is represented in Fig. 1. Out of eight cell lines tested, 2DG exerts the least cytotoxic effect on cell line originated from noncancerous immortalized cells (HEK 293, IC50:
FIG. 1. The sensitivity of various cell lines to 2-deoxyglucose (2DG) treatment. The IC50 of 2DG was calculated in various cell lines after treating with 2DG for 72 h. The colorectal cancer cell lines SW620, SW480, and GC3/Cl exhibit a lower IC50 value compared to other cell lines. The data represents the mean § SD of 3 replicates.
6.452 mM), which further supports its more selectivity toward cancer cell lines. The IC50 values of the 3 colorectal cancer cell lines were lower than that of other cancer cell lines tested. The metastatic breast cancer cells (MDA-MB-231, IC50 D 1.874 mM) are more susceptible to 2DG treatment than nonmetastatic breast cancer cells (MCF7a, IC50 D 5.891 mM). Taken together, it is clear that colorectal cancer cells are more susceptible to 2DG treatment than other cancer cells tested. Therefore, we have continued to use colorectal cancer cells (SW620) for further analysis of 2DG activity. 2DG inhibits the progression of cell cycle in colorectal cancer cell lines. The reduced viability of cancer cells after 2DG treatment could be interpreted as a result of inhibiting cell cycle progression or inducing cell death or both. Therefore, the role of 2DG in modulating the cell cycle progression was evaluated in SW620 cell line using flow cytometric analysis as described in Methods. Treatment of SW620 cells with 2DG for 18 h caused cell cycle arrest at the G0/G1 phase (Fig. 2A and 2B). The percent of cells in G0/G1 phase have significantly increased from 62.49% to 80.15% (P < 0.05; student ttest) after 2DG treatment. Simultaneously the proportion of cells in S phase has decreased from 19.11% to 7.13%.
2DG Alters the Expression of Various Cell Cycle Associated Proteins Expression levels of various proteins involved in cell cycle are critical for proper progression of cell cycle (17). Treatment with 2DG decreased the expression of various proteins associated with cell cycle progression (Fig. 3). 2DG treatment caused a downregulation of cyclin A, cyclin D1, and CDK4, whereas the levels of CDK2 and cyclin E remained unaltered.
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FIG. 3. Relative expression of various cell cycle proteins and relative density measurements. SW620 cells were treated with various doses of 2-deoxyglucose (2DG) for 18 h. The expression levels of various cell cycle associated proteins were checked by Western blot analysis followed by measuring pixel density using ImageJ software (n D 3). The figure is a representative image of 3 separate experiments. (P < 0.05; n D 3).
FIG. 2. Cell cycle analysis of SW620 cells after 2-deoxyglucose (2DG) treatment. Control SW620 cells (A) or after treatment with 20 mM 2DG for 18 h (B) were fixed and stained with PI and analyzed for DNA content using flow cytometer. The data represents mean percent of cells in a particular cell cycle phase § SD (n D 3).
Such a reduced expression of CDK4 and other cell cycle associate proteins may be responsible, in part, for the G0/G1 cell cycle arrest observed after 2DG treatment (Fig. 2).
2DG Increases the Expression of p21 and p53 Proteins The protein p21 is one of the key CDK-inhibitory proteins exerting a regulatory role in cell cycle progression. The expression of p21 can be p53-mediated or p53-independent. Treatment with 2DG increased the expression of p21 as well as p53 in SW620 cell lines analyzed by Western blot analysis (Fig. 4A). This magnitude of increase was dependent on the dose of 2DG (Fig. 4A). The kinetics of p21 and p53
expression in 2DG treated (5 mM) SW620 cells was analyzed by Western blot analysis. 2DG dependent increased expression of p21 was transient with the maximum expression occurring after 18 h of treatment (Fig. 4B). However, the protein levels of p53 increased even after 18 h of 2DG treatment and remained same for at least 48 h.
2DG Causes Apoptosis in SW620 Cells Through Intrinsic Pathway Cell cycle arrest at G0/G1 phase and the transient over expression of p21 protein may direct the cells towards apoptosis (21,22). To check whether the treatment of 2DG causes the induction of apoptosis, SW620 cells were treated with various doses of 2DG and the proportion of apoptotic cells was analyzed using TUNEL assay. 2DG treatment in SW620 cells caused an increase in TUNEL positive cells. Around 18% and 50% of cells showed TUNEL staining after treating with 10 mM and 20 mM 2DG, respectively (Fig. 5A–C). The involvement of apoptosis was further confirmed by increased cleavage of PARP after 2DG treatment (Fig. 5D). Apoptosis could occur either by intrinsic or extrinsic pathways. The
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FIG. 4. Expression of p21 and p53 after 2-deoxyglucose (2DG) treatment in SW620 cells. A: The SW620 cells were treated with various concentrations of 2DG for 48 h and amounts of p21 and p53 proteins expressed in the cell lysates were analyzed by Western blot analysis. B: Cells were treated with 5 mM of 2DG for indicated times and the amount of p21 and p53 proteins expressed were analyzed by Western blot analysis. The relative density of various protein levels normalized to b-Tubulin were plotted. The figure is a representative image of 3 separate experiments (* and #P < 0.05; n D 3).
following sets of results indicate that 2DG caused apoptosis in colon cancer cells (SW620) through intrinsic or mitochondrial pathway. Treating the SW620 cells with 2DG caused 1) increased expression of pro-apoptotic protein BAX (Fig. 6A), 2) release of cytochrome-C from the mitochondria into cytosol (Fig. 6B), and 3) loss of mitochondrial potential (Fig. 6C). The enrichment of subcellular fractions was confirmed by Western blot analysis with organelle specific marker proteins (Fig. 6D).
2DG Dependent Overexpression of p21 and p53 Is Independent of Glycolytic Inhibition 2DG is known to inhibit glycolysis. Therefore, the effect of 2DG on expression levels of p21 and p53 could be an indirect effect of 2DG due its inhibition on glycolysis or through an independent unknown mechanism. To address this, another glycolysis inhibitor, 3-bromopyruvate (3BP) was used to inhibit glycolysis (20). Similar to 2DG, 3BP is also a wellknown inhibitor of an enzyme hexokinase that is involved in glycolysis. Inhibition of glycolysis by 3BP did not increase the expression of p21 and p53 proteins (Fig. 7). This data clearly indicates that inhibiting the hexokinase enzyme by 2DG and thereby glycolysis in colorectal cancer cells may not be responsible for its effects on the protein levels of p21 or p53.
DISCUSSION The biggest drawback of current cancer chemotherapy is the extensive tissue toxicity of conventional chemotherapeutic agents (21). Therefore, preferential killing of cancer cells without significant toxicity to normal cells is an important consideration for chemotherapy. Cancer cells are metabolically more active and depend on glycolysis as an energy source for their survival. 2DG is a small molecular inhibitor of glycolysis with very good safety index (23–25). Recent studies have indicated that the glycolytic inhibition effect of 2DG alone will not explain its success in various preclinical and clinical studies in cancer (26,27). In this article, we have investigated additional mechanisms by which 2DG mediates its cytotoxic or antiproliferative actions in colorectal cancer cells. 2DG accumulates predominantly in tumors but does not harm other tissues. Despite its safety profile and preference to cancer, its success is yet to be translated to clinical application due to its high dose requirement as a single agent (65–100 mg/ Kg body weight) (8,11). However, recently, 2-DG has been tried as an effective adjuvant for chemotherapeutic agents to enhance their efficacy (28,29). Although, 2DG enhanced the efficacy of several chemotherapeutic agents, it has also lowered the effectiveness of other treatments (30). Therefore, the success of the combination therapy with 2DG may depend on the selection of the appropriate chemotherapeutic agent. We hypothesize that the understanding of molecular mechanisms
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FIG. 5. Detection of apoptosis after 2-deoxyglucose (2DG) treatment. SW620 cells were treated with PBS (A), 10 mM of 2DG (B), or 20 mM of 2DG (C) for 72 h (n D 3). The proportion of apoptotic cells were identified by TUNEL staining followed by flowcytometric analysis. D: Expression of cleaved PARP after treatment with various concentrations of 2DG for 72 h in SW620 cells. The relative density of various protein levels normalized to b-Tubulin were plotted. The figure is a representative image of 3 separate experiments (*P < 0.05; n D 3).
behind 2DG effectiveness will help in judicial selection of the combination drug for cancer treatment. In this article, we have shown that 2DG modulates the expression of various proteins involved in the regulation of cell cycle (Fig. 3). The expression of p21 protein is enhanced transiently by 2DG. In contrast, enhanced expression of p53 was continued for at least 48 h. By modulating the expression of proteins that regulate cell cycle (Fig. 3), 2DG arrested the cell cycle in colon cancer cells at G0/G1 phase. CDKs plays a critical role in the progression of cell cycle beyond G1 check point, especially CDK2 and CDK4, and are active targets for anticancer drugs (31). Although, the cell cycle was arrested at the G0/G1 phase, the level of CDK2 remained unaltered. In contrast, the protein levels of CDK4 were significantly reduced. This observation is consistent with previous reports on colorectal cancer cell lines that activity of CDK2 is dispensable for cell cycle progression and inhibition of CDK4 is sufficient to cause G0/G1 cell cycle arrest (32,33). Thus, the cell cycle arrest at the G1 phase caused by the 2DG treatment is consistent with the decreased CDK4 and Cyclin D1 levels.
In addition to cell cycle arrest, 2DG has also induced apoptosis in colon cancer cells. Apoptosis typically proceeds by either the intrinsic pathway through the release of cytochrome c from the mitochondria into cytosol to activate apoptosomes or the extrinsic pathway, in which caspases are activated directly downstream of death receptors (34,35). In colon cancer cells, 2DG induced apoptosis through intrinsic pathway involving mitochondria. This finding is consistent with previous observations that 2DG has successfully been used to sensitize cancer cells to agents that alter mitochondrial function such as mitochondrial inhibitors (oligomycin and antimycin) (36), delocalized cationic compounds (mito-Q) (36), and Bcl2 antagonists (ABT 263 and ABT 737) (36). Importantly, some of these sensitizing effects of 2DG have been shown to be independent of glucose depletion. These observations indicate that the anticancer effects of 2DG can be attributed at least in part to mechanisms other than interfering with glycolysis through hexokinase inhibition. Thus, the knowledge of the additional mechanisms of 2DG on cell cycle and mitochondrial driven apoptosis will provide
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FIG. 6. Levels of Bax, Cytochrome c, and mitochondrial potential after 2-deoxyglucose (2DG) treatment. SW620 cells were treated with different doses of 2DG and the levels of Bax (A) and Cytochrome c (B) expression were analyzed using Western blot analysis. C: The mitochondrial potential was determined using JC1 dye after 2DG treatment by analyzing the red:green fluorescence in SW620 cells. D: Cox IV and a-tubulin, which are markers for mitochondria and cytosol, respectively, were used to show the enrichment of mitochondrial and cytosolic fractions.
crucial information for selecting suitable chemotherapeutic combinations to maximize the therapeutic efficacy. 2DG is known to inhibit glycolysis. The novel pathways that are affected by 2DG (p21, p53 expression) as observed in this article, could be indirect effects of its inhibition of glycolysis or completely independent molecular events. Cancer cells predominantly depend on ATP generated by glycolysis for their energy source. 2DG inhibits hexokinase, a key enzyme in glycolysis and, thus, starves cancer cells of ATP. The effect of 2DG observed in this study could be a direct result of either ATP starvation or hexokinase inhibition. The mechanisms are dissected by physiological and pharmacological approaches (Fig. 7). To simulate the ATP starvation in cancer cells without inhibiting glycolysis, colon cancer cells were grown in media in which glucose was replaced by galactose. In the presence of galactose, the cells are unable to generate ATP using glycolysis. During glycolysis, 1 glucose molecule is converted to pyruvate and generates 2 molecules of ATP, whereas galactose enters glycolysis through galactose phosphate and generates no net ATP. Therefore, in presence of galactose, the glycolytic pathway is not inhibited, but the
production of ATP is halted for cancer cells (37). We observed that replacement of glucose in the media by galactose did not induce a significant cell death (data not shown) and also had no effect on the levels of p21 or p53 (data not shown). This observation clearly indicates that the effects of 2DG on colon cancer cells are not due to mere glycolytic ATP starvation. However, the effects could be a result of direct hexokinase inhibition by 2DG. To simulate the inhibition of glycolysis through hexokinase inhibition, the cells were treated with 3-bromopyruvate, another potent inhibitor of hexokinase. The purpose of the experiment is not to compare the efficiencies of 3BP and 2DG as anticancer drugs, but to use 3BP as a biochemical tool to inhibit hexokinase. Although both 2DG and 3BP inhibit hexokinase, only 2DG altered the levels of p21 and p53 in colorectal cell line. Taken together, the experiments with galactose and 3BP strongly suggest that the effects of 2DG on p21/p53 pathway are independent molecular events beyond ATP starvation or hexokinase inhibition. Understanding these additional mechanisms will most certainly enable us to judicially choose right combination of anti-cancer drugs to use with 2DG.
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FIG. 7. Protein levels of p21 and p53 after 3-Bromopyruvate treatment. SW620 cells were treated with different doses of 3BP and the levels of p21 and p53 expression were analyzed using Western blot analysis.
CONCLUSIONS The work proposes an additional molecular mechanism of anticancer activity of 2DG in colorectal cancer cell lines. The findings of the work also open up possibilities of novel combination therapies of agents affecting the p21/p53 protein system along with 2DG in colorectal cancer such as doxorubicin (38). Also, with the current knowledge in formulation design, these novel drug therapies could be targeted to the colon to exert maximum efficacy and reduce the overall untoward effects.
ACKNOWLEDGEMENTS We are thankful to Dr. Bhimanna Kuppast for his valuable suggestions and expert comments on the work.
FUNDING We also thank the Department of Pharmaceutical Sciences, South Dakota State University for funding the study.
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