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ORIGINAL ARTICLE
Year : 2013  |  Volume : 9  |  Issue : 4  |  Page : 686-692

Differential cytotoxicity of the glycolytic inhibitor 2-deoxy-D-glucose in isogenic cell lines varying in their p53 status


1 Division of Radiation Biosciences, Institute of Nuclear Medicine And Allied Sciences, Delhi, India
2 Department of Zoology, University of Delhi, Delhi, India

Date of Web Publication11-Feb-2014

Correspondence Address:
Bilikere S Dwarakanath
Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences Brig. SK Mazumdar Road, Delhi - 110 054
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.126484

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 > Abstract 

Context: Earlier studies have shown that cytotoxicity of glycolic inhibitor 2-deoxy-D-glucose is heterogeneous among different tumor cell lines due to a number of reasons including difference in p53 status.
Aim: To investigate the cytotoxic effects of 2-DG in isogenic cell systems that differ in their p53 status.
Material and Methods: Head and neck carcinoma cells KB and its two p53 mutants (KB68-mutation in transactivation domain (Arg/Cys) at position 68 and KB110-mutation in proline rich deoxyribonucleic acid binding domain (Glu/Gly) at position 110) were used as the model. Clonogenecity, cell proliferation, cell cycle, annexin V assay, intracellular levels of ROS and NADP+/NADPH levels were investigated as parameters for 2-DG induced cytotoxicity.
Results: Macrocolony assay showed that the cytotoxicity of 2-DG was time- and concentration-dependent. However, the sensitivity of the three cell lines were quantitatively different, with KB110 being more sensitive than the parental cell line KB and KB68. The effects of 2-DG on growth inhibition, cell cycle, and apoptosis correlated well with the changes in cell survival in these cells. A higher degree of 2-DG induced enhancement in the metabolic oxidative stress was evident in both the mutant cell lines (elevated ROS level and NADP+/NADPH ratio) suggestive of a higher degree of compromize in the antioxidant defense in the mutant cells.
Conclusions: 2-DG could be considered as a potential therapeutic agent that induces cell death (which could be linked to induced oxidative stress) selectively in tumors with p53 mutations (particularly in the proline rich region).

Keywords: 2-Deoxy-D-glucose, oxidative stress, p53, point mutations


How to cite this article:
Vibhuti A, Muralidhar K, Dwarakanath BS. Differential cytotoxicity of the glycolytic inhibitor 2-deoxy-D-glucose in isogenic cell lines varying in their p53 status. J Can Res Ther 2013;9:686-92

How to cite this URL:
Vibhuti A, Muralidhar K, Dwarakanath BS. Differential cytotoxicity of the glycolytic inhibitor 2-deoxy-D-glucose in isogenic cell lines varying in their p53 status. J Can Res Ther [serial online] 2013 [cited 2020 Oct 31];9:686-92. Available from: https://www.cancerjournal.net/text.asp?2013/9/4/686/126484


 > Introduction Top


Malignant transformation of cells is often characterized by enhanced aerobic glycolysis, the conversion of glucose into lactate even in the presence of oxygen, known as the Warburg effect. [1],[2] This characteristic metabolic pattern is an attractive target for cancer diagnosis, treatment, and prevention. 2-Deoxy-D-glucose (2-DG), an analogue of glucose and inhibitor of glycolysis competitively inhibits glucose transport (by sharing the same glucose transporters) and is phosphorylated by hexokinase (HK) to form 2-DG-6-phosphate, which is not metabolized further to any significant extent, and inhibits phosphohexoisomerase and glucose-6-phosphate dehydrogenase (G6PD) thereby reducing the output from glycolysis (adenosine triphosphate [ATP]) and the pentose phosphate pathway (PPP) (NADPH). [3],[4],[5],[6],[7] 2-DG has also been found to alter N-linked glycosylation leading to unfolded protein responses and induce changes in gene expression and phosphorylation status of proteins involved in signaling, cell cycle control, DNA repair, calcium influx, and cell death via apoptosis. [8],[9],[10] Studies on tumor-bearing animals have revealed a great deal of heterogeneity and dose dependency in the extent of growth inhibition and cure rates. [11],[12],[13],[14] The analogue has been found to be selectively toxic to a number of cancer cell lines with minimal effects in normal cells. [15],[16],[17],[18]

The TP53 is one of the most important tumor suppressor genes, often referred to as "the guardian of the genome." In unstressed cells, the p53 protein is maintained at very low levels, regulated by the proteasome mediated degradation. In response to various intracellular and extracellular stresses, such as damages to DNA integrity, hypoxia, and oncoprotein expression, p53 is rapidly stabilized and activated primarily due to its posttranslational modifications. The activated p53 mainly functions as a sequence-specific DNA-binding transcription factor to regulate a large number of target genes that mediate cell cycle arrest, apoptosis, senescence, differentiation, DNA repair, inhibition of angiogenesis and metastasis, and other activities including prevention of cancer development. Regulation of mitochondrial respiration by p53 is due to the identification of its transcriptional target protein named synthesis of cytochrome C oxidase 2 (SCO2), a key regulator of the cytochrome c oxidase complex. [19] A p53-inducible gene named TIGAR (TP53-induced glycolysis and apoptosis regulator), has been shown to function to lower the intracellular levels of fructose-2,6-bisphosphate (Fru-2,6-P2), a substrate that promotes glycolysis by activation of 6-phospho-1-kinase, a key enzyme in the glycolytic pathway. [20] Enhanced TIGAR expression lowers Fru-2,6-phosphate levels, thereby slowing glycolysis and directing glucose to the alternative PPP. Since PPP is the major source of NADPH required for the scavenging of reactive oxygen species (ROS) by reduced glutathione (GSH), induction of this pathway by TIGAR can result in the decrease of ROS levels and lower cellular sensitivity to ROS-associated apoptosis. Loss of p53-induced TIGAR activity in cancers has been suggested to contribute to the Warburg effect; enhanced glycolysis. Further wild-type p53 downregulates the expression of phosphoglycerate mutase (PGM), an enzyme in the glycolytic pathway, [21] and thus altered p53 function resulting in increased PGM expression can also enhance glycolysis. These roles of p53 in regulating metabolic pathways has been identified recently, which regulates the balance between oxidative phosphorylation and glycolysis and disruption of this balance is associated "with mutations in p53 and oncogenic transformation." Mutations in p53 contribute to resistance and/or sensitivity of many cancers to cellular response to various chemotherapeutic agents would also differ with respect to specific mutation. Present study aims at the elucidation of the functional significance of two point mutations specifically found in patients with HNSCC (human neck squamous cell carcinoma), which occur in the proline rich transactivation domain (Glu/Gly) and DNA binding domain (Arg/Cys) at positions 68 and 110, respectively (http://www.iarc.fr/p53). Since, both these mutations have been reported in precancerous lesions, they may also have a role in manifestation and progression of HNSCC. Since PRD is known to regulate apoptosis, mutations in PRD regulate the response of many chemotherapeutic agents.

Here, we have investigated the effects of the glycolytic inhibitor, 2-DG on head and neck carcinoma cell line KB and two isogenic lines with miss sense mutations in p53 at 68 and 110 positions in the proline rich region. Results show that the mutant cell lines are relatively more sensitive to 2-DG induced cytotoxicity suggesting thereby that inhibition of glycolysis may be an attractive strategy for the treatment of head and neck cancer having p53 mutations at least in the proline rich region.


 > Materials and Methods Top


Cell culture

The head and neck squamous carcinoma cell line KB and its p53 mutants (KB 68 and KB 110) were maintained as monolayer cultures in DMEM supplemented with 10% fetal bovine serum (FBS), HEPES (10 mM) and antibiotics (30 μg/ml penicillin G, 50 μg/ml streptomycin, and 2 μg/ml nystatin) in a humidified CO 2 incubator at 37°C (5% CO 2 , 95% air). All cell lines were routinely subcultured (twice a week) using 0.05% trypsin in 0.02% ethylene-diamine-tetraacetic acid (EDTA) and reseeded in fresh medium. All experiments were carried out in exponentially growing cells. DMEM (high glucose) and FBS were purchased from SIGMA chemicals (SIGMA, USA). Cells used in this study are established cell lines and P53 mutant clones (KB68 and KB110) were provided by Prof Uttam Pati laboratory (Department Of Biotechnology, JNU).

Experimental procedure

Exponentially growing cells were allowed to attach and grow (i.e., 24 h after seeding before treatments) and all treatments were carried out under suboptimal growth (liquid holding) conditions by replacing the growth medium with Hanks balanced salts solution (HBSS) containing 5 mM glucose. Cells were treated with 2-DG for 4 or 24 h and 2-DG removed by washing and replacing with fresh growth medium and allowed to grow to study various response parameters.

Clonogenic cell survival

Exponentially growing cells (150-600 cells, depending on the treatment and plating efficiency) were plated in 60 mm Petri dishes in triplicates. Following treatments, cells were incubated (12-14 days, depending on their doubling time) at 37°C in a humidified CO 2 ( 5%) conditions for colony formation. Colonies were fixed in methanol and stained with crystal violet (0.1% in 70% methanol). Colonies with more than 50 cells were counted.

Cell proliferation and cell cycle distribution

Exponentially growing cells were seeded at 1 × 10 5 cells/Petri dish of 35-mm diameter for cell proliferation studies. Immediately after treatment cells were harvested at every 24 up to 72 h by trypsinization and counted using hemocytometer. For determining the proliferation, (Nt/N0) was calculated, where, N0 is the total number of cells at the time of treatment and Nt is the total number of cells at time t. Progression of cells through different phases of cell cycle was also measured flow cytometricaly along with growth kinetics. Both floating and attached cells were counted and fixed in 80% chilled ethanol for cell cycle analysis. Cellular DNA content was measured in ethanol fixed cells using the intercalating DNA fluorochrome, propidium iodide (PI) as described earlier. [22] The cells (0.5-1 × 10 6 ) were washed with Phosphate Buffer Saline (PBS) after removing ethanol and treated with RNase A (200 μg/ml) for 30 min at 37°C. Subsequently cells were stained with PI (25 μg/ml) for 15 min at room temperature. Measurement were made with an argon laser-based flowcytometer (FACS-Calibur Becton Dickinson San Jose, CA, USA) using the argon laser (488 nm) for excitation. Distribution of cells in different phases of cell cycle was calculated from the frequency distribution of DNAcontent by using the Mod fit Program (Variety Software, CA, USA).

Annexin V binding

Translocation of the phosphatidylserine (PS) from inner to the outer side of the plasma membrane is one of the manifestations of apoptosis. Therefore, externalization of PS was studied at different time intervals after treatment by annexin V-fluorescein isothiocyanate (FITC) kit (Sigma, USA) according to manufacturer's instructions. Briefly, cells (1 × 10 5 ) were resuspended in 200 μl of binding buffer (10 mM HEPES/NaOH, pH 7.4, 10 mM NaCl, 2.5 mM CaCl2) and 5 μl annexin V-FITC and 5 μl PI (50 μg/ml) were added. After 15 min of incubation at room temperature in the dark, samples were analyzed flow cytometrically. The percentage of annexin V-positive and -negative cells were estimated by applying appropriate gates and using regional statistics analysis facility provided in the Cell Quest Software (Becton-Dickenson, San Jose, CA, USA).

reactive oxygen species measurement

Intracellular ROS generation was measured by flow cytometry using the H2DCF-DA dye according to the procedure described earlier. [23] Briefly, following treatment at specific time point, cells were washed with PBS. Stock solution of H2DCFDA (1 mg/ml) was prepared in methanol and stored at 4°C in the dark. Working concentration of H2DCFDA (10 μg/ml) was prepared in a buffer solution consisting of 1 mM CaCl2, 1 mM MgSO4, and 5 mM glucose/dextrose in PBS, pH 7.2. Freshly prepared working solution of H2DCFDA was added to the samples and incubated in the dark at 37°C for 30 min. Cells were then washed with PBS, scraped gently, resuspended in PBS and transferred to flow tubes and kept ice cold until measurement by flow cytometry. Data was acquired and analyzed using the CELL Quest program (Becton Dickinson).

NADPH and NADP + measurements

Following the treatments, cells were washed with PBS twice and scrape-harvested in PBS at 4°C. After centrifugation at 300g for 5 min, cell pellets were resuspended in 200 μl of extraction buffer containing 0.1 M Tris-HCl, pH 8.0, 0.01 M EDTA, and 0.05% (v/v) Triton X-100. The cell suspension was sonicated using a Vibra Cell sonicator (Sonics) at a duty cycle of 30% for 2 min at 30-s intervals on ice. The solution was centrifuged at 5500g for 5 min. The supernatants were collected and analyzed immediately for NADP + and NADPH. [24] Briefly, an aliquot (50 μl) of the extract was incubated with 950 μl of extraction buffer at 37°C for 5 min and an absorbance measurement was taken at 340 nm. This reading measures the total amount of NADPH and NADH in the sample (A1). Another 50 μl aliquot of the extract was preincubated at 37°C for 5 min in a reaction mixture containing 5.0 IU of G6PD, 0.1 M Tris-HCl, pH 8.0, 0.01 M MgCl 2 , and 0.05% (v/v) Triton X-100. The reaction was initiated by the addition of 5 mM glucose 6-phosphate. After incubation of the mixture at 37°C for 5 min, absorbance measurement was taken at 340 nm. This reaction converted NADP + to NADPH (A2). Finally, a 50 μl aliquot of the extract was preincubated at 25°C for 5 min in a reaction mixture containing 5.0 IU of glutathione reductase, 0.1 M phosphate buffer, pH 7.6, 0.05 mM EDTA, and 0.05% (v/v) Triton X-100. The reaction was initiated by the addition of glutathione disulfide (GSSG, 5 mM) to convert NADPH to NADP + . The absorbance of the mixtures at 340 nm was determined (A3). Subtraction of A3 from A1 represents the total amount of NADPH in the sample. The total amount of NADP + was calculated by subtracting the A1 from A2. Results were obtained by comparison with a standard curve.

Statistical analysis

Mean values of data were analyzed for significance by standard Student's t-test. Statistical analysis between multiple groups was examined by using analysis of variance. A of P ≤ 0.05 was considered significant.


 > Results Top


Cytotoxicity of 2-DG were studied by investigating its effects on clonogenicity and cell proliferation in exponentially growing conditions. In these experiments, cells were treated with 2-DG for 4 and 24 h followed by washing with HBSS and plated to study various end points.

Clonogenicity

The clonogenic potential assessed by macrocolony assay revealed marked differences between the three cell lines. The plating efficiency of KBwt was 50%; while it was 33% for KB68 and 26% for KB110. Macrocolony assay performed to assess effects on clonogenic survival showed that the cytotoxicity of 2-DG was both time- and concentration-dependent, with both the mutant cell lines showing a higher degree of sensitivity [Figure 1]. In the wild-type cells, a 20% reduction in survival was observed at 10 mM 2-DG and 4 h exposure, which marginally increased to 26% with 24 h exposure. These values were 30% and 36%, respectively, for KB68 cells, while they were 34% and 56%, respectively, for KB110 cells [Figure 1].
Figure 1: Concentration and time dependent effects of 2-DG observed on the clonogenicity of wild-type and mutant head and neck carcinoma cells. Cells (150-600; depending on the treatment and plating efficiency of cell lines) were seeded in triplicate and exposed to 2-DG. Following treatment, cells were incubated (10-14 days) and stained with 1% crystal violet. Colonies with more than 50 cells were counted. Data presented are mean ± 1SD of 10-12 observations from four independent experiments. *P < 0.05 vs. respective control

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Cell proliferation

The effect on cell proliferation were investigated at 10 mM 2-DG by comparing the cumulative growth at the end of 72 h after exposure to 2-DG. While the KB wild-type and KB110 showed little change in the growth for a 4 h exposure, nearly 40% inhibition in the growth was evident in KB68 cells [Figure 2]. However, at 24 h exposure, all three cell lines showed significant growth inhibition, with KB68 being more sensitive than the other two [Figure 2]. Interestingly, a 30% and 45% decrease in the extent of growth was noted in untreated KB68 and KB110 cells as compared with the wild-type cells [Figure 2].
Figure 2: 2-DG (10 mM) induced growth inhibition in KBwt, KB68 and KB110 cells. Cells treated with 2-DG for 4 and 24 h, respectively, were harvested at regular time intervals (24-72 h) after the treatment by trypsinization and resuspended in PBS for enumeration of numbers using a hemocytometer. Data presented are mean ± 1 SD of 10-12 observations from four independent experiments. *P < 0.05 vs respective control, #P < 0.05 vs similar treatment of wild-type

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In order to understand the reasons for the growth inhibition (reduction in cell numbers; [Figure 2]) observed, we examined for 2-DG (10 mM, 24 h) induced cell cycle perturbation by cytoflurometric measurements of cellular DNA content at various time intervals. Flow cytometric DNA histograms presented in [Figure 3] did not show significant changes induced by 2-DG in the distribution of cells in different phases of the cell cycle in KBwt as well as KB68 and KB110 cells. However, DNA histograms at 72 h post-2-DG treatment in KB110 cells revealed a large fraction of degenerating/dead cells, possibly suggestive of apoptotic and/or necrotic death. Annexin-V binding assay (a measure of apoptotic cells due to externalization of phosphotidyl serine) carried out at 72 h after exposure of cells to 2-DG showed 50-60% increase in the fraction of apoptotic cells in KB68 and KB110, while <5% apoptotic cells were found to be apoptotic in KBwt [Figure 4].
Figure 3: 2-DG (10 mM, 24 h) induced degeneration of cells selectively in KB110 cells. Representative histograms from one typical experiment showing treatment-induced changes in DNA content distributions observed at different time intervals are presented for all three cell types (in KB Wt, KB68, and KB110) cells. Ethanol fixed cells were stained with PI (0.5 ìg/ml) and analyzed by flow cytometry. Similar observations were made in 3 (n=3) independent experiments

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Figure 4: 2-DG (10 mM, 24 h) induced apoptosis in malignant KB68 and KB110 cells. After 72 h posttreatment cells were incubated with fluorescein isothiocyanate conjugated annexin V, counter labeled with PI (5 μg/ml) and analyzed flow cytometrically for FITC (green) and PI (red) fluorescence. The percentage of annexin V-positive were estimated by applying appropriate gates and using regional statistics analysis facility provided in the CELL Quest Software. Data presented are mean ± 1SD of 10-12 observations from four independent experiments. *P < 0.05 vs. respective control, #P < 0.05 vs similar treatment of wild-type

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Oxidative stress

In order to understand the reason for apoptosis especially in 2-DG treated mutant cell lines, oxidative stress was measured, as it is well known that during 2-DG treatment the ability of cancer cells to metabolize hydroperoxide is compromised, this is based on the fact that accumulation of 2-DG-6-phosphate can disturb the pentose cycle that allows regeneration of NADPH from NADP+, leading to metabolic oxidative stress, while p53 also plays an important role in the maintenance of physiological ROS in living cells by regulating the flux between mitochondrial respiration and glycolysis. Since oxidative stress is a strong inducer of apoptosis, we investigated the effects of 2-DG (10 mM, 24 h) on the oxidative stress in wild-type and mutant cells by analyzing the levels of NADP+/NADPH and levels of ROS (DCFDA assay). Under the present experimental conditions, 2-DG significantly decreased the NADPH levels in all the three cell lines leading to enhanced NADP+/NADPH ratio, although the extent of increase significantly higher in both the mutant cell lines (1.8- to 2.5-folds) compared with the wild-type cells (1.5-fold) [Table 1]. This increase in NADP+/NADPH ratio indicates compromised redox balance suggestive of metabolic oxidative stress correlated with an increase in the ROS levels measured in these cells [Figure 5]. Interestingly, the endogenous ROS levels were 2- to 3-folds higher in both the mutant cell lines as compared with the wild-type cells [Figure 5], suggestive of the role of p53 linked maintenance of (metabolic) oxidative stress.
Figure 5: 2-DG (10 mM, 24 h) induced generation of reactive oxygen species in KB, KB68, and KB110 cells. After 24 h posttreatment cells were incubated with DCFDA (10 μM) at 37°C for 30 min in the dark and analyzed flow cytometrically. Green fluorescence due to dichlorofluorescein (DCF) were collected on the FL1 on a log scale. The data analysis was carried out using the data acquisition program CELL Quest (Becton-Dickinson, San Jose, CA, USA). Mean fluorescence intensity was calculated after correction for auto-fluorescence. Data presented are mean ± 1SD of 10-12 observations from four independent experiments. *P < 0.05 vs respective control, #P < 0.05 vs. control of wild-type

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Table 1: Alteration in NADPH levels (mM) and NADP+/NADPH ratio in wt, 68 and 110 cells treated with 2-DG (10 mM, 24h)

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 > Discussion Top


The glucose antimetabolite, 2-DG, a competitive inhibitor of glucose transport and glucose phosphorylation by HK, is known to selectively inhibit glycolytic energy (ATP) production [25],[26],[27] and therefore energy-dependent cellular processes including maintenance, growth and cellular repair/recovery processes. [28],[29],[30] Several studies have examined the effects of 2-DG as a primary therapeutic agent in providing local tumor control in a number of murine and rodent tumor models [13],[14],[31],[32] as well as xenografted human tumors. [33]

Tumor suppressor p53 plays a role in energy metabolism by regulating metabolic processes. [34] p53 stimulates oxidative phosphorylation after sensing decrease of ATP through up regulation of the SCO2 gene that encodes a copper chaperone protein required for the assembly of mitochondrial cytochrome c oxidase (complex IV) [19],[35] as well as transcriptional activation of subunit I of cytochrome coxidase. [36] Furthermore, p53 activates TP53-induced glycolysis and the apoptosis regulator (TIGAR), which functions to direct glucose to the PPP, as well as G6PD, a glycolytic enzyme that catalyzes a rate-limiting step in the PPP. [37],[38] The increase in PPP results in the stimulation of nucleotide synthesis and production of NADPH, which is an important component of the antioxidant defense system. [20] In addition to the role of p53 in the regulation of mitochondrial respiration, p53 inhibits glycolysis by repressing the transcription of GLUT1, GLUT4 genes that encode glucose transporters [39] and the PGM gene that encodes a glycolytic enzyme responsible for the rearrangement of phosphoglycerate. [21] Mutation (s) leading to alterations in the functioning of p53 is expected to disturb the p53 mediated regulation of a balance between oxidative phosphorylation and glycolysis, as well as the redox balance. Therefore, such mutations are likely to influence the viability and proliferation of cells as well as the response to inhibition of glycolysis (caused by inhibitors like 2-DG). Both the mutant cell lines (KB 68 and KB 110) showed enhanced oxidative stress in the form of elevated ROS levels [Figure 5] and increase in NADP+/NADPH ratio [Table 1] suggesting alterations in the redox balance possibly due to disturbed balance in the p53 mediated regulation of mitochondrial function (including ROS generation) and glycolysis linked antioxidant defense. This enhanced oxidative stress resulted in a reduction in the clonogenic efficiency of the mutant cells, where 20-30% reduction in the plating efficiency was noted [Figure 1].

Inhibition of glycolysis by 2-DG is expected to cause both energy depletion and oxidative stress, which would depend both on the concentration of 2-DG (in fact the ration of glucose to 2-DG) as well as duration of exposure compromising viability as well as proliferation. The degree of susceptibility would, however, depend on the extent of dependency on glycolysis. While mutant p53 is likely to enhance glycolysis, it may also affect the oxidative stress induced cell death by the intrinsic apoptotic pathway, which is generally facilitated by wild-type p53. The cytotoxicity of 2-DG was clearly higher in both the p53 mutant cells (KB68 and KB110), particularly at higher concentration and longer exposure time as seen by the loss of clonogenicity [Figure 1] and growth inhibition [Figure 2]. This was accompanied by enhanced apoptosis as revealed by the presence of hypo diploid population [Figure 3] and annexin V binding [Figure 4].

2-DG-induced cytotoxicity may be due to metabolic oxidative stress in cancer cells. [40],[41],[42] After entry into the cells 2-deoxyglucose 6-phosphate (the product of 2-DG from the HK reaction) is not a substrate for the second pentose cycle enzyme, which also regenerates one molecule of NADPH, 6-phosphogluconate dehydrogenase. [40] Thus, a second molecule of NADPH would not be generated from the pentose phosphate cycle when 2-deoxyglucose 6-phosphate was the substrate, relative to glucose 6-phosphate. In this regard 2-DG metabolism in the PPP would be expected to limit the regeneration of NADPH by 50%, relative to glucose metabolism, because glucose would regenerate two molecules of NADPH, relative to one molecule being regenerated from the metabolism of 2-DG. 2-DG probably negatively impacts on H 2 O 2 detoxification in two ways. First, 2-DG, by virtue of inhibiting glycolysis after the step catalyzed by HK, will decrease pyruvate formation and pyruvate has been shown to be a good H2O 2 scavenger via a deacetylation reaction that results in the formation of acetic acid, water, and CO 2 . [43] Second, 2-DG would be expected to limit the amount of NADPH that can be regenerated from the PPP (relative to glucose) because it is apparently not a substrate for 6-phosphogluconate dehydrogenase. Because NADPH is a required cofactor for glutathione- and thioredoxin-dependent peroxidase, this property of 2-DG would also be expected to contribute to sensitization to hydroperoxide toxicity.

Glycolytic inhibitor-induced metabolic oxidative stress is mediated either by an enhanced intracellular superoxide production (because of increased mitochondrial metabolism) or due to decrease in the level of pyruvate and/or NADPH. The oxidative stress induced by 2-DG was relatively more pronounced in both the mutant cells as seen by the higher NADP+/NADPH ratio and ROS levels [Table 1] and [Figure 5] suggesting a relatively higher dependency on glycolysis in the mutant cells, in line with the other observations.

At basal levels, p53 is required to maintain a normal basal transcription of antioxidant genes, SESN1 (mammalian sestrin homolog), SESN2 and GPX1 (glutathione peroxidase-1) [27] and antioxidant enzyme AIF (apoptosis-inducing factor). [44] Mutations in p53 has been shown to result in a significant decrease in the basal levels of transcriptions of SESN1, SESN2, and GPX1 without affecting the expression of pro-oxidant genes BAX, NQO1, and PUMA. [45] This leads to an increase in ROS, which was observed in the present study where higher endogenous ROS levels were evident in both the mutant cell lines [Figure 5].


 > Conclusion Top


Taken together, results from the present study suggest that the fate of head and neck cancer cells is strongly influenced by the positions of p53 mutations and exposure and concentration of glycolytic inhibitor 2-DG. Our result support the proposition that head and neck carcinoma cells, which bear mutations in p53 at its proline rich regions at positions 68 and 110, are more susceptible to glycolytic inhibitor-induced cytotoxicity than the parental cells possibly due to enhanced metabolic oxidative stress mediated by 2-DG linked to mutations in p53. Therefore, glycolytic inhibitors like 2-DG, which cause cellular death, could be partly due to oxidative stress selectively in head and neck carcinoma cells with p53 mutations (particularly in the proline-rich region like 68 and 110 positions studied here) and merits consideration as potential therapeutic agents.

 
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    Figures

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