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ORIGINAL ARTICLE
Year : 2009  |  Volume : 5  |  Issue : 9  |  Page : 57-60

Modulation of cellular radiation responses by 2-deoxy-D-glucose and other glycolytic inhibitors: Implications for cancer therapy


Department of Biophysics, National Institute of Mental Health and Neuro Sciences, Bangalore, India

Date of Web Publication21-Aug-2009

Correspondence Address:
Vijay K Kalia
Department of Neurophysiology, National Institute of Mental Health and Neuro Sciences, Bangalore - 560 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.55145

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 > Abstract 

Background: 2-Deoxy-D-glucose (2-DG), a glycolytic inhibitor, was observed earlier to increase DNA, chromosomal, and cellular damage in tumor cells, by inhibiting energy-dependent repair processes. Lonidamine (LND) selectively inhibits glycolysis in cancer cells. It damages the condensed mitochondria in these cells, impairing thereby the activity of hexokinase (predominantly attached to the outer mitochondrial membranes). It inhibits repair of radiation-induced potentially lethal cellular damage in HeLa and Chinese hamster (HA-1) cells. However, other than a preliminary study on human glioma (BMG-1) cells in this laboratory, the effects of LND on radiation-induced cytogenetic damage have not been reported earlier.
Aims: These studies were carried out to investigate the effects of LND and 2-DG on cell proliferation, viability, and radiation response in the same human glioma cell line, under identical conditions. The respective drug concentrations were selected on the basis of earlier studies.
Materials and Methods: Human glioma (U373MG) cells were grown in the presence of LND or 2-DG for 2 days. Proliferation response and viability of U373MG human glioma cells were studied by cell counts and uptake of trypan blue dye. Radiosensitization (increase in micronuclei formation) was studied after short-term (4 h postirradiation) drug treatments.
Observations: Both the drugs (1) inhibited proliferation response in a concentration-dependent manner; (2) did not induce micronuclei formation in the unirradiated cells; and (3) significantly increased radiation-induced micronuclei formation at nontoxic concentrations.
Conclusions: These data suggest that the short-term presence of either lonidamine or 2-DG-at clinically relevant and nontoxic concentrations-could increase the treatment response of malignant gliomas at optimum radiation doses, reducing thereby the side effects of radiotherapy.

Keywords: Cytotoxicity, glioma cells, lonidamine, radiation damage, 2-deoxy-D-glucose


How to cite this article:
Kalia VK, Prabhakara S, Narayanan V. Modulation of cellular radiation responses by 2-deoxy-D-glucose and other glycolytic inhibitors: Implications for cancer therapy. J Can Res Ther 2009;5, Suppl S1:57-60

How to cite this URL:
Kalia VK, Prabhakara S, Narayanan V. Modulation of cellular radiation responses by 2-deoxy-D-glucose and other glycolytic inhibitors: Implications for cancer therapy. J Can Res Ther [serial online] 2009 [cited 2019 May 19];5:57-60. Available from: http://www.cancerjournal.net/text.asp?2009/5/9/57/55145


 > Introduction Top


Malignant tumors, such as grade III/IV astrocytomas, have significantly increased glycolysis as the source of cellular metabolic energy. [1] Several investigations demonstrated that 2-deoxy-D-glucose (2-DG, a glucose analog) inhibited glycolysis (and consequently, the ATP flow below certain critical threshold levels), leading to enhancement of radiation-induced DNA, chromosomal, and cellular damage in different types of cancer cells. [2],[3],[4],[5] In contrast, radiation-induced chromosomal damage was considerably reduced in PHA-stimulated peripheral human leukocytes and murine bone-marrow cells. [6],[7],[8] Subsequently, several other anticancer agents with diverse mechanisms of action, such as misonidazole, SR-2508, hyperthermia, N -ethylmaleimide (NEM), were also found to act partly through inhibition of energy metabolism or/and repair of radiation damage in cancer cells. Energy-linked modulation of repair processes was, therefore, proposed as a promising approach for optimizing cancer therapy. [9] LND is also known to be a selective inhibitor of glycolysis in cancer cells.[10] It damages the condensed mitochondria in tumor cells, [11] inhibiting thereby the activity of hexokinase, which is predominantly attached to the outer membranes of mitochondria. Short-term (2-6h) treatment with LND inhibited radiation-induced potentially lethal damage repair (PLDR) in HeLa and Chinese hamster (HA-1) cells.[12],[13] Preliminary studies in a human glioma (BMG-1) cell line showed, for the first time, that short-term LND treatment also increased radiation-induced micronucleus formation [14] which correlates with enhanced cell death. [15] Similar effects of 2-DG had already been observed in BMG-1 cells under these experimental conditions. [5],[8],[16] The present studies were carried out to verify the effects of 2-DG and LND-at their respective, clinically relevant and nontoxic concentrations-on radiation response under identical experimental conditions, in the same human glioma cell line. Data show that both the drugs significantly increased radiation-induced cytogenetic damage in U373MG cells.


 > Materials and Methods Top


Lonidamine was provided by F. Angelini Research Institute (Rome, Italy) and 2-DG was obtained from Serva Feinbiochemica (Heidelberg, Germany). All the other reagents were analytical grade (Qualigens Fine Chemicals, Bangalore, India). The malignant glioma cell line (U373MG) was obtained from the National Centre for Cell Science (NCCS), Pune (India). The culture medium (MEM, Eagle's) and trypsin were obtained from HiMedia (Mumbai, India). MEM contained nonessential amino acids, l-glutamine, Earle's salts, and 2.2g/l sodium bicarbonate, and was supplemented with 0.11g/l sodium pyruvate (Sigma Chemicals) and antibiotics. The growth medium contained 10% fetal bovine serum (Life Technologies, USA). Cultures were maintained in 25cm 2 plastic flasks (Orange Scientific, Belgium). Every 5 days, near-confluent cultures were trypsinized (rinsed once and incubated with fresh trypsin 0.1%, prewarmed to 37C) and subcultured, with an initial inoculum of 3 10 5 cells. The experimental cultures were set up in six-well plates, with 5 10 4 cells in each well.

Lonidamine was dissolved in dimethyl sulfoxide (DMSO) to prepare the stock solution (10mM) and stored at 4C for up to 4 weeks. The final concentration of DMSO in the growth medium, in all the LND-treated as well as control cultures, was maintained at 0.1%. The stock solution of 2-DG (100mM) was prepared in sterile double-distilled water. The control cultures contained sterile double-distilled water in the growth medium (5%, corresponding to 1:20 dilution for 5mM 2-DG treatment). Dilutions from the stock solutions were made just before adding drugs to the experimental cultures. The concentrations and protocols for drug treatments were selected on the basis of earlier studies. [7],[8],[9],[14],[16] Drug solutions (or solvents alone, for drug-free controls) were added to different wells, 3 days after setting up the cultures. For the study of proliferation response, cultures were grown further in continuous presence of drugs for 2 days. Cultures were trypsinized, resuspended in MEM, and pooled together with floating cells in the supernatant medium. Cells were then centrifuged (800-1,000rpm) for 10min, stained with trypan blue dye (0.5% solution in 1 phosphate-buffered saline), and counted twice under a hemocytometer chamber (Rohem, India). All the cells, which excluded trypan blue (viable) as well as those which took up the dye (nonviable), were accounted for the determination of cell growth. The percentage of nonviable cells was calculated on the basis of total number of (viable plus nonviable) cells. For the study of radiation damage, drugs were added in a fresh growth medium and cultures were exposed to gamma irradiation ≈60min after the addition of drugs (in the Department of Radiation Physics, Kidwai Memorial Institute of Oncology, situated close by) with Theratron 780C (Atomic Energy of Canada Ltd.; focus distance 80cm, field size 35 x 35cm, dose rate 0.62Gy/min, total dose 2Gy) at room temperature. After 4-h incubation at 37C, the drugs were removed by decanting the medium. Cultures were rinsed with, and grown further in the normal growth medium without DMSO. After 24h of growth, including postirradiation incubation with drugs, the cultures were harvested by trypsinization. After centrifugation, the medium was decanted; cells were resuspended in a small volume of residual supernatant, fixed, and left overnight in Cornoy's fixative (methanol:acetic acid 3:1, at 4C). After two changes of the freshly prepared fixative, the cells were resuspended; slides were prepared by air drying and stained with 0.002% acridine orange (AO), prepared in Sorenson's buffer (0.1M, pH 6.8). At least 1,000 cells from each slide were scanned under an inverted fluorescence microscope (Olympus, IX 70, Japan), using 20 objective (at a total magnification of 200). Cells with micronuclei were scored, using the criteria of Countryman and Heddle. [17] Statistical significance of the differences between various treatments was estimated by ANOVA and/or an appropriate " t" test as applicable using the SPSS program, version 10.0 of Windows 2000.


 > Results Top


A continuous presence of 0.1, 0.15, and 0.2 mM LND for 2 days significantly reduced the cell proliferation response as compared to the drug-free controls-in a concentration-dependent manner-to 71.23%, 58.97%, and 49.76%, respectively ( P = 0.000; Dunnett's " t" [two-sided] test, [Figure 1]a). In contrast, 2-DG at 0.1 mM, had no effect on cell growth. However, 1 and 5mM 2-DG significantly reduced the proliferation response to 84.78% and 68.22%, respectively, as compared to the control cultures ( P = 0.000; Dunnett's " t" [two-sided] test, [Figure 1]b). Cell viability, as measured by trypan blue uptake, was not affected by 2-DG treatments, and the lowest concentration of LND (0.1mM). Higher concentrations of LND, however, significantly increased the frequencies of nonviable cells from 2.34% in controls to 13.67% and 17.11% ( P = 0.000; Dunnett's " t" [two-sided] test, [Figure 1]a and b). The presence of LND (0.1mM) and 2-DG (5mM) for 4 h after gamma irradiation (2Gy) significantly increased the frequency of cells with micronuclei (M-Fraction; P = 0.000; 2 Gy vs. 2 Gy + 0.1mM LND and P = 0.001; 2Gy vs. 2Gy + 5.0mM 2-DG; independent samples' " t"-test, [Figure 2]a and b). The sensitization enhancement ratio (SER) after LND and 2-DG treatment was 1.35 and 1.29, respectively.


 > Discussion Top


In the present study, effects of both LND and 2-DG on cell proliferation, viability, and radiation response have been investigated in the same malignant human glioma (U373MG) cell line. A continuous presence of 0.1mM 2-DG for 2 days had no effects on cell growth, whereas LND-induced inhibition at this concentration was higher than that of 1mM 2-DG. Loss of cell viability (as measured by the uptake of trypan blue dye) was observed only at higher concentrations of LND (>0.1mM). Comparative effects of these drugs on proliferation response, viability, and other cellular characteristics in glioma cell lines, therefore, need to be investigated in detail. Short-term treatment with 2-DG (2-4h, mostly at 5mM concentration, equimolar with glucose in the medium) has earlier been reported to significantly increase radiation-induced micronuclei formation, or inhibit PLDR in BMG-1 and other types of cancer cells. [2],[8],[16] Lonidamine treatment (≈0.033-0.156 mM for 2-7h) inhibited X-ray-induced PLDR in HeLa and Chinese hamster (HA-1) cells, albeit at relatively high (8-12Gy) radiation doses. [12,13] Earlier studies from this laboratory in BMG-1 glioma cells [14] showed for the first time that LND treatment (20g/ml ≈0.062mM, 2h) inhibited PLDR induced by gamma irradiation at the clinically relevant dose of 2Gy ( P = 0.0017, 2Gy vs. 2Gy + 0.062mM LND; two samples' " t"-test data reproduced in [Figure 3]). Radiation-induced micronuclei formation was also increased by LND treatment (SER at 24h = 1.52, data not shown). The present observations, therefore, support the earlier suggestion [14] that the enhancement of radiation-induced cytogenetic damage is an important mechanism, contributing to the radiosensitization of cancer cells by LND.

2-DG has been tolerated very well in combination with radiotherapy in an earlier clinical trial on malignant brain tumors [18] and further trials are in progress (proceedings of the present symposium). LND has also been used as an adjuvant in radiotherapy of brain tumors, [19] and several experimental multiple-chemotherapy protocols for other kind of tumors. It has shown a better treatment response, without additional toxicities such as myelosuppression.[20] Present data, therefore, strongly suggest that further preclinical studies should be undertaken to investigate the mechanisms of action as well as potential of glycolytic inhibitors-including 2-DG and LND-to selectively modulate the radiation response of treatment-resistant tumors, including malignant gliomas.


 > Acknowledgements Top


This work was supported by research grants from BRNS, DAE (no. 2002/37/21/BRNS) Government of India, and NIMHANS. V.K.K. acknowledges Neurochemistry and Neurovirology departments for all the laboratory facilities, for the DAE project work. Gamma irradiations were carried out in the Department of Radiation Physics, Kidwai Memorial Institute of Oncology (KMIO), Bangalore. Lonidamine was a free gift by F. Angelini Research Institute, Italy. The paper was presented at the Symposium on "Applications of 2-deoxy-D-glucose in the management of cancer," Institute of Nuclear Medicine and Allied Sciences, Delhi, India, November 8-10, 2006. Travel of V.K.K. and S.P. was partly supported by the symposium.

 
 > References Top

1.Oudard S, Arvelo F, Miccoli L, Apiou F, Dutrillaux AM, Poisson M, et al. High glycolysis in gliomas despite low hexokinase transcription and activity correlated to chromosome 10 loss. Br J Cancer 1996;74:839-45.  Back to cited text no. 1
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2.Jain VK, Purohit SC, Pohlit W. Optimization of cancer therapy: Part I. Inhibition of repair of X-ray induced potentially lethal damage by 2-Deoxy-D-Glucose in Ehrlich ascites tumour cells. Indian J Exp Biol 1977;15:711-3.  Back to cited text no. 2
    
3.Jain VK, Kalia VK, Sharma R, Maharajan V, Menon M. Effects of 2-Deoxy-D-Glucose on glycolysis, proliferation kinetics and radiation response of human cancer cells. Int J Radiat Oncol Biol Phys 1985;11:943-50.  Back to cited text no. 3
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4.Verma A, Sharma R, Jain VK. Energetics of DNA repair in UV irradiated peripheral blood leukocytes from chronic myeloid leukemia patients. Photochem Photobiol 1982;36: 627-32.  Back to cited text no. 4
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5.Dwarkanath BS, Jain VK. Energy linked modifications of the radiation response in a human cerebral glioma cell line. Int J Radiat Oncol Biol Phys 1989;17:1033-40.  Back to cited text no. 5
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6.Jain VK, Kalia VK, Gopinath PM, Naqvi S, Kucheria K. Optimization of cancer therapy: Part III- Effects of combining 2-Deoxy-D-Glucose treatment with gamma irradiation on normal mice. Indian J Exp Biol 1979;17:1320-5.  Back to cited text no. 6
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7.Kalia VK, Jain VK, Otto FJ. Optimization of cancer therapy: Part IV. Effects of 2-Deoxy-D-Glucose on radiation induced chromosomal damage in PHA stimulated peripheral human leukocytes. Indian J Exp Biol 1982;20:884-8.  Back to cited text no. 7
    
8.Kalia VK, Devi NK. Differential modification of radiation damage in 5-bromo-2-deoxy-uridine sensitized human glioma cells and PHA-stimulated peripheral leukocytes by 2- Deoxy-D-Glucose . Indian J Exp Biol 1994;32:637-42.  Back to cited text no. 8
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9.Kalia VK, Dwarkanath BS, Jain VK. Optmization of tumour radiotherapy by energy linked modulation of repair processes. AMPI Medical Physics Bulletin 1987;12:94-112.  Back to cited text no. 9
    
10.Floridi A, Paggi MG, Marcante ML, Silvestrini B, Caputo A, De Martino C. Lonidamine, a selective inhibitor of aerobic glycolysis of murine tumor cells. J Natl Cancer Inst 1981;66:497-9.  Back to cited text no. 10
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11.De Martino C, Battelli T, Paggi MG, Nista A, Marcante ML, D'Atri S, et al. Effects of Lonidamine on murine and human tumor cells in vitro : A Morphological and Biochemical study . Oncology 1984; 41:15-29.  Back to cited text no. 11
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12.Kim JH, Kim SH, He SQ, Alfieri AA, Young CW. Potentiation of radition effects on multicellular tumour spheroids (MTS) of Hela cells by Lonidamine. Int J Radiat Oncol Biol Phys 1989;16:1277-80 .  Back to cited text no. 12
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13. Hahn GM, van Kersen I, Silvestrini B. Inhibition of the recovery from potentially lethal damage by Lonidamine. Br J Cancer 1984;50:657-60.  Back to cited text no. 13
    
14.Kalia VK, Narayanan V. Optimizing treatment of brain tumors: Effects of Lonidamine and 5-bromo-2-deoxy-uridine on radiation response of human glioma cells. NIMHANS J 1995;13:133-9.  Back to cited text no. 14
    
15.Midander J, Revesz L. The frequency of micronuclei as a measure of survival in irradiated cell population. Int J Radiat Biol 1980;38:237-42.  Back to cited text no. 15
    
16.Kalia VK. Optimizing radiation therapy of brain tumors by combination of 5-bromo-2-deoxy-uridine and 2-deoxy-D-glucose. Indian J Med Res 1999;109:182-7.  Back to cited text no. 16
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17.Countryman PI, Heddle JA. The Production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutat Res 1976;41:321-32.  Back to cited text no. 17
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18.Mohanti BK, Rath GK, Anantha N, Kannan V, Das BS, Chandramouli BA, et al. Improving cancer radiotherapy with 2-Deoxy-D-Glucose: Phase I/II clinical trials on human cerebral gliomas. Int J Radiat Oncol Biol Phy 1996;35:103-11.  Back to cited text no. 18
    
19.Schiffer D, Sales S, Soffietti R. Lonidamine in Maliganant Brain Tumors. Semin Oncol 1991;18:38-41.  Back to cited text no. 19
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20.Teicher BA. Lonidamine: In Vitro / in vivo correlations. Eur J Cancer 1994;30A:1411-3.  Back to cited text no. 20
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    Figures

  [Figure 1], [Figure 2], [Figure 3]


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