|Year : 2009 | Volume
| Issue : 9 | Page : 67-73
Short-term exposure of multicellular tumor spheroids of a human glioma cell line to the glycolytic inhibitor 2-deoxy-D-glucose is more toxic than continuous exposure
Divya Khaitan, Sudhir Chandna, S Bilikere Dwarakanath
Department of Biocybernetics, Institute of Nuclear Medicine and Allied Sciences, Brig. S K Mazumdar Marg, New Delhi, India
|Date of Web Publication||21-Aug-2009|
S Bilikere Dwarakanath
Division of Biocybernetics, Institute of Nuclear Medicine and Allied Sciences, Brig. S K Mazumdar Road, Timarpur, New Delhi -110 054
Source of Support: None, Conflict of Interest: None
The glycolytic inhibitor 2-deoxy-D-glucose (2-DG) has been used as a therapeutic agent and as an adjuvant in cancer therapy with either weekly fractions of the treatment or daily administration. While the weekly fraction has often been found to be nontoxic and effective, other treatment regimes are tolerated to a relatively lesser extent. It was therefore, considered worthwhile to investigate the efficacy of short- and long-term exposure of tumor cells to 2-DG under the controlled conditions.
Seven-day-old MTS were exposed to 2-DG (5 mM, equimolar to glucose concentration in media) for different time intervals (30 min to 24 h) trypsinized and plated for clonogenecity. Alternatively, spheroids were grown either continuously in the presence of 2-DG or were treated with 2-DG for 2 h (short-term exposure) and grown in 2-DG-free media for 21 days and assessed for spheroid growth, cell viability, apoptosis, cytogenetic damage, mitochondrial status, and oxidative stress.
Exposure of spheroids to 2-DG for 2-4 h induced 30% cell death (SF 0.70) while, a 24-h exposure resulted in only a marginal decrease in clonogenecity (SF 0.95). Furthermore, the spheroids disintegrated completely by 28 days in the case of 2-h exposure to 2-DG, while spheroids grown continuously in the presence of 2-DG repopulated. The cytotoxicity following short-term exposure of MTS to 2-DG was primarily due to the induction of apoptosis revealed by morphological features as well as flow cytometric analysis of the DNA content. Interestingly however, cytogenetic damage (micronuclei induction) was observed in spheroids that were continuously exposed to 2-DG. Short-term exposure to 2-DG resulted in a significant increase in ROS levels and a reduction in the levels of unoxidized cardiolipin as measured by NAO suggesting the involvement of mitochondria leakiness leading to oxidative stress which, could be responsible for apoptotic cell death observed under these conditions. However, continuous exposure to 2-DG resulted in a moderate level of oxidative stress leading to the genomic instability. Preliminary studies also show that spheroids exposed continuously to 2-DG result in the development of resistance to certain chemotherapeutic drugs which could be correlated with elevated levels of mdr1. The present results suggest that a persistent down-regulation of glycolysis (as seen here with continuous exposure to 2-DG) could activate prosurvival responses besides inducing moderate levels of oxidative stress resulting in the development of resistance against therapeutic agents.
Keywords: Glioma cell line, spheroids, 2-deoxy-D-glucose, cytotoxicity, apoptosis, necrosis
|How to cite this article:|
Khaitan D, Chandna S, Dwarakanath S B. Short-term exposure of multicellular tumor spheroids of a human glioma cell line to the glycolytic inhibitor 2-deoxy-D-glucose is more toxic than continuous exposure. J Can Res Ther 2009;5, Suppl S1:67-73
|How to cite this URL:|
Khaitan D, Chandna S, Dwarakanath S B. Short-term exposure of multicellular tumor spheroids of a human glioma cell line to the glycolytic inhibitor 2-deoxy-D-glucose is more toxic than continuous exposure. J Can Res Ther [serial online] 2009 [cited 2019 May 21];5:67-73. Available from: http://www.cancerjournal.net/text.asp?2009/5/9/67/55147
| > Introduction|| |
Tumors generally show elevated glucose-dependent aerobic and anaerobic metabolic pathways due to both hypoxia-induced and hypoxia independent enhancement in the glycolytic pathways, , which correlate directly with the degree of malignancy and inversely with the prognosis. , 2-Deoxy-D- glucose (2-DG), an analog of glucose and inhibitor of glycolysis, retards the growth of tumor cells both in vitro and in vivo and induces cell death in certain types of tumors. ,,,,, However, the undesirable normal tissue toxicity mainly in the form of central nervous system disturbances and diaphoresis observed during the daily administration of large amounts of 2-DG required to achieve tumor growth inhibition limited its use as a primary therapeutic agent in clinics.  Therefore, it has been suggested that 2-DG could be useful as an adjuvant for improving therapeutic responses, by short-term administration at appropriate time intervals with radiation  and chemotherapeutic drugs. ,
Recent studies using genetically manipulated cell lines of human origin (tumorigenic and nontumorigenic) have focused on the mechanisms of growth inhibition and cell death as well as identification of tumors that would be susceptible to local control using glycolytic inhibitors like 2-DG. , Most of these studies have employed monolayer cultures that have limitations in predicting the in vivo response of tumors as well as providing information on the mechanisms underlying the tumor responses to therapeutics including 2-DG. The limitation can be overcome by the use of spheroid models that permit growth and functional studies of diverse normal and malignant tissues, mimicking the in vivo conditions that provide insights into tumor physiology, and response to therapeutic agents. Results of the present studies clearly show that short-term exposure of spheroids to 2-DG is more toxic as compared to continuous exposure. Further, the long-term exposure of spheroids to 2-DG appears to induce adaptation by way of protection against glucodeprivation-induced apoptosis.
| > Meterials and Methods|| |
The cerebral glioma cell line (BMG-1; wild-type p53) was established in Bangalore, India.  Stock cultures were maintained in the exponentially growing state by passaging twice weekly in DMEM containing 10 mM HEPES and antibiotics supplemented with 5% fetal bovine serum.
A detailed procedure for establishing and growing BMG-1 spheroids has been reported.  Briefly, spheroids were grown by inoculating 1 × 10 6 viable cells of exponentially growing BMG-1 monolayer cells in nonadherent 90-mm Petri dishes in 10 ml DMEM supplemented with 5% fetal calf serum, antibiotics, and 10 mM HEPES.
2-Deoxy-D-glucose (2-DG) at a concentration equimolar with glucose (5 mM) was added in spheroids. After 2 h, 2-DG was removed; spheroids were washed and reseeded to study different parameters. 2-DG was present throughout the experimental time in the 2-DG continuous treatment groups.
Spheroid growth measurements
The measurement of spheroid growth was carried out as reported earlier.  Briefly, images of spheroids were obtained using the image analysis system consisting of Olympus BX 60 fluorescence microscope and Grundig FA87 monochrome CCD camera. The geometric mean radius r = 1/2ο d 1 d 2 was calculated by measuring two orthogonal diameters ( d 1 and d 2) using the line morphometry function, and volume was computed using the formula V = 4/3οr 3 .
Cell proliferation and viability
Following different treatments and time points, spheroids were trypsinized and cells counted with the help of hemocytometer. The number of viable cells was determined on the basis of the trypan blue dye (0.4% dissolved in PBS) exclusion assay. The fraction of live cells was computed.
Air-dried slides of acetic acid-methanol fixed cells were stained with a DNA-specific fluorochrome, diamidino-2-phenylindole dihydrochloride, and DAPI, and approximately 1,000 cells were analyzed from duplicate slides. Data were analyzed by obtaining integrated values of micronuclei frequency and normalizing the values with respect to cell numbers as described earlier.  The frequency of cells with micronuclei, called the M-fraction (MF) was calculated as: MF (%) = N m / N t × 100, where N m is the number of cells with micronuclei and N t is the total number of cells analyzed.
Cell cycle distribution
Flow cytometric measurements were performed with 80% ethanol-fixed cells stored at least overnight at 4°C using intercalating DNA fluorochrome propidium iodide (PI) as described earlier.  Data were acquired with the help of FACS Calibur flow cytometer (Becton-Dickinson & Co., USA) using the CellQuest software (version 3.0.1; Becton Dickinson & Co., USA) and cell cycle distribution was analyzed using ModFit LT software (version 2.0; Verity Software House, Inc., USA).
Following treatment at different time intervals, spheroids were washed with PBS and dissociated into single cells by trypsinization and counted using a hemocytometer. An appropriate number of cells were plated in 60-mm Petri dishes with complete media containing 10% FCS. Plates were incubated at 37°C in a humidified incubator (5% CO 2 and 95% air) till colonies were formed (7 days). Methanol-fixed colonies were stained with 1% crystal violet and colonies containing more than 50 cells were scored. The plating efficiency (PE) was calculated as the fraction of plated cells forming macrocolonies (%). Surviving fraction was calculated as SF = PE Treated /PE Control .
Estimation of glucose utilization
Spheroids were incubated in HBSS or HBSS and 2-DG for 2 h. The amount of glucose remaining unused and the lactate produced were estimated in the buffer using enzymatic assays. Glucose was measured using the reducing sugar method.  Briefly, a mixture of equal volumes of the incubating medium (HBSS) and alkaline copper sulfate was incubated at 900°C for 10min, followed by the addition of phosphmolybidic acid at room temperature (~27°C). OD of the resulting chromogen was measured at 540 nm. The number of viable cells in spheroids was counted and glucose consumed was normalized with respect to number of viable cells.
Determination of intracellular redox levels
Intracellular redox levels were measured according to the procedure described earlier using the fluorescent dye H 2 DCFDA. , Briefly, following treatment at regular intervals of time, unfixed spheroids were washed and incubated with PBS (Ca 2+ , Mg 2+ , H 2 DCFDA [10 mg/ml] and 5 mM glucose), for half an hour at 37°C followed by trypsinization, washing, and resuspension in PBS with Ca 2+ , Mg 2+ , and glucose and incubated on ice until acquisition by the flow cytometer.
Flow cytometric analysis of mitochondrial mass and activity
Spheroids were stained with one of the mitochondrial fluorescent dyes NAO and Rh123, essentially according to the procedure described earlier. , Briefly, the unfixed spheroids were washed and incubated in PBS (with Ca 2+ , Mg 2+ ), glucose, and NAO (10 mM, 10min at 25°C) or Rh123 (5 mg/ml, 30min at 37°C), followed by trypsinization washing and resuspension in PBS (with Ca 2+ , Mg 2+ , and glucose) and incubated on ice until acquisition by the flow cytometer.
| > Results|| |
Glucose utilization and lactate production
Our previous results have shown that like many other spheroid models, endogenous levels of glycolysis (glucose utilization and lactate production) are higher in BMG-1 spheroids as compared to monolayers.  Therefore, we investigated the effects of the glucose antimetabolite 2-DG on the glucose consumption following incubation of spheroids with 2-DG (5 mM 2-DG; 2-DG/G = 1) for short intervals (2 h) or following continuous exposure up to 4 days. (Untreated spheroids and spheroids treated with 2-DG for 2 h required media change for their maintenance and growth.) The glucose consumption rate of the untreated spheroids and spheroids treated with 2-DG for 2 h decreased gradually with time in culture [Figure 1], although no significant difference was noted between the consumption rates between these two groups. This decrease could be either due to a decrease in pH or due to a large fraction of cells becoming quiescent under these conditions. However, the rate of glucose consumption in spheroids continuously exposed to 2-DG was significantly lower (50%) than control, which continued further, till 96 h posttreatment [Figure 1].
Clonogenic cell survival
The three-dimensional growth patterns of spheroids inhibit the drug penetration. Therefore, studies were carried out to determine the toxicity of 2-DG in the 7-day-old spheroids using clonogenic assays by trypsinizing and plating spheroidal cells following exposure to 2-DG for different time intervals. The toxicity profile was biphasic, with maximum toxicity observed at 2- to 4-h exposure to 2-DG (SF = 0.70 and 0.67 respectively) [Figure 2]. Interestingly, further incubation of the spheroids with 2-DG enhanced the clonogenicity in a time-dependent manner with the SF value reaching 0.93 at 24 h [Figure 2].
Growth of spheroids and viability of spheroidal cells
To understand the mechanisms of cell death during short-term (2 h) exposure and development of resistance during long- term exposure to 2-DG, spheroid growth, cell viability, and the nature of cell death were investigated. Seven-day-old spheroids were treated either for 2 h (short-term) or continuously (long- term) for 7-28 days, and spheroids radius was measured using image analysis, while viability analyzed using trypan blue dye exclusion after trypsinizing the spheroids until they disintegrated (approx. 28 days following the start of treatment).
Under the present experimental conditions, the growth of spheroids treated with 2-DG for 2 h was nearly identical to untreated spheroids till the seventh day with similar spheroid volumes [Figure 3]. This was followed by a gradual disintegration evidenced by the presence of small clumps, numerous free-floating single cells, and cellular debris resulting in a decrease in the volume and cell viability, reaching a size that was less than 20% of the untreated spheroids at 28 days and did not repopulate thereafter [Figure 3]. In contrast, spheroids treated continuously with 2-DG showed initial growth retardation and signs of disintegration for the first 7 days, but recovered thereafter as evidenced by an increase in the volume [Figure 3] and viability [Figure 4]. These spheroids grew rapidly once they reached a critical volume (0.05- 0.06mm 3 ) and disintegrated after reaching a large size similar to untreated spheroids as reported earlier. 
Interphase death in the form of apoptosis and/or necrosis and mitotic death (linked to cytogenetic damage) are primary modes of cell death induced by a variety of cytotoxic agents.
Induction of apoptosis was studied by the morphological analysis of cells stained with Hoechst-33342, and DNA analysis by flow cytometry. A gradual increase in the apoptotic cells was observed when spheroids were treated with 2-DG for 2 h grown in the 2-DG-free medium thereafter. At 28 days posttreatment, more than 65% of the cells were apoptotic [Figure 5]. On the other hand, continuous exposure of 2-DG increased the apoptotic cells from 2% to 17% at 14 days followed by a decrease in the frequency of apoptotic cells (10%) thereafter [Figure 5]. This observation correlated well with the appearance of a hypodiploid peak (indicative of apoptotic cells) and fraction of degenerating cells observed in the DNA histograms by flow cytometry (data not shown). Interestingly, we observed a significant fraction of micronucleated cells in spheroids treated continuously with 2-DG, suggesting the induction of cytogenetic damage under these conditions.
The kinetics of micronuclei induction clearly showed that continuous exposure of spheroids to 2-DG led to micronucleation in more than 40% of the cells at 14 days, followed by a decrease reaching 10% at 28 days, a value still higher than the untreated spheroids [Figure 6]. The kinetics of micronuclei induction was somewhat similar in spheroids treated with 2-DG for 2 h, although, the extent was significantly lower as compared to the continuous exposure [Figure 6]. These results suggest that both short- and long-term exposure of spheroids to 2-DG results in genetic instability, albeit to different extents, but eventually lead to adaptation of cells during continuous exposure.
ROS generation and mitochondrial status
Glucodeprivation, including treatment with 2-DG, has been shown to induce oxidative stress and cause cytotoxicity.  Therefore, we investigated differences in the induction of oxidative stress (using H 2 DCFDA) as one of the contributing factors for the differential cytotoxicity observed with short- and long-term exposure of spheroids to 2-DG. Spheroids treated continuously with 2-DG did not show an increase in ROS during the 7-21 days of observation, while a short duration of exposure (2 h) showed a two- to threefold gradual increase in the oxidative stress which persisted till 7-21 days posttreatment [Figure 7]a. Mitochondria are the major site of ROS production and alterations in the induction of ROS are generally associated with perturbations in mitochondrial mass and function, which are manifested as a change in number of mitochondria per cell or dissipation of the transmembrane potential. Therefore, mitochondrial mass and activity of spheroidal cells were analyzed following short- and long-term exposure of spheroids to 2-DG. Significant changes in the mitochondrial mass and activity could not be observed following the continuous treatment [Figure 7]b and c]. Although no significant change in mitochondrial mass and activity was observed at 7 days following a 2-h exposure to 2-DG, a two-to threefold decrease was observed at 14 days posttreatment which continued till 21 days posttreatment [[Figure 7]b and c]. These results correlate well with the generation of ROS observed and suggest that leakiness of ROS from mitochondria is mainly responsible for the enhanced ROS observed and is also one of the main contributing factors for the enhanced delayed cell death observed following a short exposure (2 h) of spheroids to 2-DG.
| > Discussion|| |
Extrinsic signals such as death-inducing ligands as well as intrinsic signals like macromolecular damage (for example, DNA damage), generation of ROS, metabolic catastrophy, etc., cause cell death through multiple mechanisms. However, exposure of cells to the same perturbing agent can elicit diametrically opposite responses under different conditions. Such responses have important implications for the design of therapy using such agents. In the present studies, the glycolytic inhibitor, 2-DG was indeed found to elicit profound death in spheroids when treated for short intervals (2-4 h) as against an adaptive not-toxic response when treated continuously for 7-21 days. Tumors display a high rate of glucose uptake and glycolysis under aerobic conditions depends on the glycolytic flux to maintain the cellular levels of ATP and metabolism. , Available evidence suggests that tumor cells of different types can be markedly sensitized to death receptor-mediated apoptosis either by glucose deprivation or by an inhibition of glucose metabolism with 2-DG. , However, the exact mechanism of sensitization is not yet clear. Since spheroids closely mimic the tumor micromilieu as compared to their monolayer counterparts, , studies obtained using spheroids may provide a chance to better understand the mechanism of cell death by 2-DG. Results of the present study demonstrate that a brief exposure of spheroids to 2-DG (2-4 h) induced progressive cell death with 30% cell death (SF 0.70) after 7 days, and the spheroids disintegrated completely by 28 days posttreatment with morphological features of apoptosis [Figure 3] and [Figure 4]. This increase in cell death could be due to changes in other metabolic parameters, as glucose deprivation has been shown to be a potent inducer of apoptosis in several c-Myc overexpressing cell lines.  Furthermore, a decrease in the constitutive regeneration of NADPH by the inhibition of glycolysis can alter the redox state in the cells that make them more susceptible to apoptosis.  Our results clearly show that short- term exposure of 2-DG enhances the oxidative stress [Figure 7]a due to leakiness of ROS from the damage to mitochondria [Figure 7]b and c. However, it appears that the cells can indeed overcome this oxidative stress by inhibiting the oxidative phosphorylation, and in turn block the ROS accumulation leading to a steady state of oxidative stress as seen with continuous exposure of spheroids to 2-DG [Figure 7]a. In vivo  as well as in vitro , studies have revealed that sensitivity of tumor cells to TNF-a is increased under conditions of reduced glucose metabolism thereby enhancing cell death. Earlier studies have clearly demonstrated that 7-day-old spheroids show enhanced glycolysis, and express an elevated level of c-Myc and TNF-a  thereby making spheroidal cells more susceptible to glucose deprivation-induced apoptosis. Further glucodeprivation in TNF-a-induced cell death of fibrosarcoma cells has been shown to be associated with increase production in reactive oxygen species arising from the metabolism of glutamine that sensitizes the cells to death receptor-mediated apoptosis.  Cytotoxicity of therapeutic agents like radiation and anti-cancer drugs is associated with two mechanistically and morphologically distinct forms of cell death that contribute to the loss of survival. One, the programmed cell death (apoptosis), characterized by nuclear condensation and DNA degradation, which is induced by both extrinsic as well as intrinsic pathways. The other is mitotic death, linked to the cytogenetic damage, which is expressed as chromosomal aberrations in the metaphase and manifests in the form of micronuclei formation in the post mitotic daughter cells that arise from the residual DNA damage, following induction and repair of DNA lesions.  The cytotoxicity by 2-DG was primarily due to the enhancement of apoptosis in spheroids following a 2-h exposure of 2-DG [Figure 5], while cytogenetic damage was relatively higher in spheroids following continuous exposure to 2-DG [Figure 6]. The induction of micronuclei seen following short and continuous exposure of spheroids to 2-DG clearly suggests that the glucodeprivation-induced oxidative stress (or metabolic oxidative stress) induces a significant level of DNA damage, which has not been observed in any of the monolayer cells investigated. , One of the reasons for this could be the synergism of glucodeprivation-mediated oxidative stress with TNF-mediated oxidative stress operating only in spheroids. Therefore, from the therapeutic perspective, an intermittent administration of 2-DG in tumors may enhance therapeutic gain by inducing both mitotic and interphase death, while continuous administration may in fact compromise the efficacy. The differential induction of apoptosis and cytogenetic damage (micronuclei) following short-term and continuous exposure of spheroids to 2-DG is consistent with the idea that these processes are regulated by different proteins and depend on different signaling pathways which may not be mutually exclusive. Alternatively, the continuous exposure of 2-DG could result in a reduced steady state of glycolysis (adaptation) and thereby facilitate the glycolytic enzymes to perform alternative tasks, like regulation of gene expression,  transport of nucleotides in synaptic vesicles,  and binding to specific tRNAs,  particularly related to, antiapoptotic cell-protective functions. GAPDH may also interact with cytoskeleton  and microtubules,  thereby protecting the cell body from apoptotic blebbing which in some instances may be a causative event of apoptosis. 
The present results therefore clearly show that a sustained down-regulation of glycolysis in 3-D systems of tumor cells like MTS and in vivo tumors may result in an active self-protective reaction of the cell leading to cell survival in at least a fraction of the cells and repopulation. Irrespective of the mechanisms underlying such a type of response, these results suggest that therapeutic gain can be enhanced by an intermittent (well-spaced) administration of metabolic inhibitors like 2-DG to tumors either alone or in combination with radiation or chemotherapeutic drugs, rather then continuous or daily administrations. Although, the earlier clinical studies with daily administration of 2-DG were discontinued mainly due to CNS disturbances and toxicity to other organs,  the present study shows that it may not even be beneficial for obtaining local tumor control. On the other hand, protocols designed with 2-DG administration well spaced between two fractions, like the hypofractionated treatment with weekly fractions of 2-DG + radiation in the clinical trials of malignant gliomas reported earlier, , may provide better local tumor control, besides reducing normal tissue toxicity.
| > Acknowledgements|| |
This work was supported by grants (INM-280 and INM-301) from DRDO, Government of India. Authors wish to thank Professor Viney Jain for his invaluable suggestions during the course of this study.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]