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Tumour cell culture survival following glucose and
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glutamine deprivation at typical physiological
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concentrations
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Edward Henry Mathews, PhDa, B. André Stander, PhDb,
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Annie M. Joubert, PhDb, and Leon Liebenberg, PhDa,*
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a
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Suite No 91, Private Bag X30, Lynnwood Ridge 0040, South Africa;
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and consultants to TEMM International (Pty) Ltd, and MCI (Pty)Ltd.
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[email protected]; [email protected]
Centre for Research and Continued Engineering Development, North-West University,
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b
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Pretoria, Pretoria 0001, South Africa.
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[email protected]; [email protected]
Department of Physiology, School of Medicine, Faculty of Health Sciences, University of
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RUNNING HEAD: Glucose deprivation for cancer control?
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* Corresponding author: Tel.: +27 12 809 0995; Fax: +27 86 661 8408
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E-mail: [email protected] (L. Liebenberg)
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WORD COUNT: 4949
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NUMBER OF FIGURES (AND TABLES): 7
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NUMBER OF REFERENCES: 37
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ABSTRACT
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Objective: Most glucose (and glutamine) deprivation studies of cancer cell cultures focus on total depletion,
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and are conducted over 24 h or longer. It is difficult to extrapolate findings from such experiments to practical
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anti-glycolytic treatments such as with insulin-inhibiting diets (with 10% - 50% carbohydrate dietary
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restriction) or with isolated limb perfusion (ILP) therapy (which usually lasts around 90 min). We aimed to
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obtain experimental data on the effect of partial deprivation of D-glucose and L-glutamine (to typical
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physiological concentrations) during 0 – 6 h exposures of HeLa cells.
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Methods: HeLa cells were treated for 0 to 6 h with 6 mM D-glucose and 1 mM L-glutamine (normal in vivo
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conditions), 3 mM D-glucose and 0.5 mM L-glutamine (severe hypoglycaemic conditions), and 0 mM
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D-glucose
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to investigate morphological changes.
and 0 mM L-glutamine (‘starvation’). PlasDIC and phase contrast light microscopy were employed
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Results: Reduction of glucose levels from 6 mM to 3 mM (and glutamine levels from 1 mM to 0.5 mM)
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brings about cancer cell survival of 73% after 2 h exposure and 63% after 4 h exposure. Reducing glucose
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levels from 6 mM to 0 mM (and glutamine levels from 1 mM to 0 mM) for 4 h resulted in 53% cell survival.
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Conclusion: These data reveal that glucose (and glutamine) deprivation to typical physiological
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concentrations result in significant cancer cell killing after as little as 2 hours. This supports the possibility of
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combining anti-glycolytic treatment, such as a carbohydrate-restricted diet, with chemotherapeutics for
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enhanced cancer cell killing.
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Keywords: Glucose deprivation; HeLa cell survival and morphology; Metabolic cancer control
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3
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Introduction
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Around 90% of cancer-associated deaths can be ascribed to highly glycolytic cancers and metastases
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(HGCM) [1]. In vitro tests have shown that increased glucose consumption in HGCM ensures that the
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metabolically inflexible cancer cells are more susceptible to glucose deprivation-induced cytotoxicity and
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oxidative stress than non-transformed cells [2-5].
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Inverse HGCM comorbidity in people with certain complex disorders could provide in vivo proof of the
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beneficial effect of low blood glucose (BG) microenvironments [6]. The typical adverse response of
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chemotherapeutics on HGCM in rodents (with low BG microenvironments) but not in their human
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counterparts (with their high BG microenvironments) provides further in vivo evidence of the beneficial
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effects of a low BG microenvironment [7].
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Recent evidence also links chronic hyperglycaemia to increased risk of most cancers [8]. Diabetogenic
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glucose concentrations ( ≈ 11 mM) compared to physiological ones ( ≈ 5.5. mM) lead to altered expression of
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genes that promote cell proliferation, migration and adhesion in tumour cells lines from several organs [9].
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Adding insulin to the high-glucose medium further enhances proliferation rates by 20-40% [9] and promotes
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activation of the tumourogenic PI3K pathway [10]. Many cancer cells express insulin receptors (IR) and
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show hyperactivation of the insulin growth factor (IGF)-IR [8,10].
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Early evidence also suggests that the anti-diabetic drug metformin is associated with lower risk of cancer
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[8,10,11]. Metformin decreases basal glucose by suppressing hepatic gluconeogenesis and glycogenolysis and
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by increasing glucose uptake in muscle tissue [8]. The lower circulating glucose levels lead to improved
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insulin sensitivity and decreased insulin levels, which are thought to diminish cancer growth [12,13].
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Further in vivo proof of the benefits of low BG levels is provided by the fact that calorie restriction in animal
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models and in humans may be responsible, directly or indirectly, for their significantly prolonged survival
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compared to normally fed control animals or humans [5,9,15]. This inhibition of tumourigenesis is possibly
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due to reduced levels of IGF-1 [10,15]; stimulation of autophagy, mitophagy and the AMPK pathway [16];
4
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and mTOR inhibition and its reduction of inflammatory cytokines [17]. Severe caloric restriction also lowers
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basal metabolic rates with a resultant decrease of mutagenic oxidative stress [18].
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Whilst the high demand for carbon in proliferating tumour cells and associated fibroblasts is provided by
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glucose metabolism [19], the high demand for nitrogen is obtained from glutamine metabolism [5,11]. Co-
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regulation of pathways that govern glucose and glutamine uptake and utilization is therefore expected [20]. It
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has been shown that glucose deprivation indeed leads to a marked reduction in glutamine uptake [21]. Also,
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some HGCM have been shown to be totally dependent on glucose, not glutamine, for energy production and
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survival [22].
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Glucose thus appears at the top of cancer cell (and cancer-associated fibroblast) metabolic hierarchy,
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followed by glutamine. Therefore, if cancer cells and associated fibroblasts can be deprived of glucose, the
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cancer cells would not only be less able to metabolise glutamine but would also be deprived of de novo
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produced lactate, fatty acids and ketone bodies. However, if simultaneous deprivation of glucose and
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glutamine can be performed, cancer cells would be dealt a severe metabolic blow. Valid predictive models for
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highly glycolytic cancers exposed to glucose (and glutamine) deprivation would be helpful in establishing
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appropriate in vivo glucose (and glutamine) concentrations to bring about maximum cancer cell killing with
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minimal ‘innocent bystander’ effects.
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Most researchers show the effect of total glucose deprivation (i.e., ‘starvation’) on cancer cell survival.
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Typically, cancer cell lines are cultured in 25 mM glucose medium and then subjected to 0 mM glucose
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medium for 24 h [2,4]. It is difficult to extrapolate from here to more typical physiological blood glucose
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(BG) environments, such as 3 mM or 6 mM glucose [19], and for shorter deprivation periods (such as 2 h or 4
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h). This may be important when translating in vitro findings to practical metabolic therapies including
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insulin-inhibiting diets [14], and ex vivo (extracorporeal) anti-glycolytic treatments such as with isolated limb
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perfusion (ILP) therapy of soft-tissue carcinoma that lasts in the vicinity of 90 min [23].
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This paper intends to partially address the lack of such in vitro data. We investigate the effect of short-term
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exposure (2 to 6 h) of HeLa cells to varying D-glucose and L-glutamine concentrations that are similar to
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typical physiological values.
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Materials and Methods
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Cell line
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The effect of combined D-glucose and L-glutamine deprivation was conducted on HeLa (human epithelial
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cervix carcinoma) cells. The HeLa cell line was purchased though Sterilab Services (Pty) Ltd, Johannesburg,
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South Africa, from the American Tissue Culture Collection (ATCC), Maryland, USA.
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General reagents
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D-Glucose, L-glutamine
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as well as high D-glucose (25.52 mM, 4500 mg/L), L-glutamine (4 mM) and sodium pyruvate (1 mM, 110
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mg/L) containing DMEM, bicarbonate, L-glutamine, D-glucose, Trypsin-EDTA, crystal violet, NaCl, KCl,
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KH2PO4 and Na2HPO4 were supplied by Sigma Chemical Co. (St. Louis, MO, USA). Heat-inactivated fetal
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bovine serum (FBS), sterile cell culture flasks and plates were obtained though Sterilab Services
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(Johannesburg, South Africa). Penicillin, streptomycin and fungizone were purchased from Highveld
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Biological (Pty) Ltd. (Sandringham, South Africa).
and sodium pyruvate-free Dulbecco’s Minimum Essential Medium Eagle (DMEM)
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General cell culture procedures
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Cells were grown and maintained in 25 cm2 tissue culture flasks in a humidified atmosphere at 37°C, 5% CO2
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in a Forma Scientific water-jacketed incubator (Ohio, United States of America). HeLa cells were cultured in
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DMEM with 25.52 mM D-glucose, 4 mM L-glutamine, and 1 mM sodium pyruvate supplemented with 10%
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heat-inactivated FBS (56°C, 30 min), 100 U/ml penicillin G, 100 µg/ml streptomycin and fungizone
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(250 µg/l).
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Cells were harvested by tripsinization and counted by making use of a haemocytometer as described by
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Freshney (2008). Suspended cells (20 µl) were mixed with 80 µl FBS and 100 µl trypan blue to provide a
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concentration of cells with a 10-times dilution factor. Dead cells absorb the dye and are consequently stained
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blue, which are then left uncounted. The number of viable cells per mL was determined by:
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Cells/ mL = Average count of viable cells in the corner squares × dilution factor × 104
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For experiments, cells were seeded in 6-well (60 000 cells per well, 500 µ L/well) tissue culture plates, or in
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25 cm2 tissue culture flasks (750 000 cells per flask). Cells were incubated for 24 hours after seeding to allow
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for attachment after which medium was removed; cells were rinsed twice with FBS and subsequently exposed
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to the various experimental conditions.
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Morphology: Light microscopy via polarization-optical differential interference contrast
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(PlasDIC) and phase contrast
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Polarization-optical differential interference contrast (PlasDIC) is a polarization-optical transmitted light
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differential interference contrast method from Zeiss where linearly polarized light is only generated after the
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objective lens [24]. PlasDIC and phase contrast light microscopy were employed to recognize and display
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morphological changes in relation to other cells in order to gain insight on the effects that various exposures
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have on the cells.
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Cell Survival Assay - Colony formation
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Cells were seeded at 60 000 cells per well in 6-well plates and cultured overnight to allow for attachment.
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After cell attachment, the medium was discarded and the cells were rinsed twice with FBS. Cells were
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exposed to the following conditions for 2 hours, 4 hours and 6 hours:
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Control: DMEM with 25.52 mM D-glucose, 4 mM L-glutamine, and 1 mM sodium pyruvate
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supplemented with 10% heat-inactivated FBS (56°C, 30 min), 100 U/ml penicillin G,
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100 µg/ml streptomycin and fungizone (250 µg/l).
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Experimental
Condition
1:
DMEM
with
6
mM
D-glucose,
1
mM
L-glutamine,
and
160
1 mM sodium pyruvate supplemented with 10% heat-inactivated FBS (56°C, 30 min),
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100 U/ml penicillin G, 100 µg/ml streptomycin and fungizone (250 µg/l).
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Experimental Condition 2: DMEM with 3 mM D-glucose, 0.5 mM L-glutamine, and 1 mM sodium
163
pyruvate supplemented with 10% heat-inactivated FBS (56°C, 30 min), 100 U/ml penicillin G,
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100 µg/ml streptomycin and fungizone (250 µg/l).
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Experimental
Condition
3:
DMEM
with
0
mM
D-glucose,
0
mM
L-glutamine,
and
166
1 mM sodium pyruvate supplemented with 10% heat-inactivated FBS (56°C, 30 min),
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100 U/ml penicillin G, 100 µg/ml streptomycin and fungizone (250 µg/l).
168
Experimental
Condition
4:
DMEM
with
6
mM
D-glucose,
1
mM
L-glutamine,
and
169
0 mM sodium pyruvate supplemented with 10% heat-inactivated FBS (56°C, 30 min),
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100 U/ml penicillin G, 100 µg/ml streptomycin and fungizone (250 µg/l).
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Experimental Condition 5: DMEM with 3 mM D-glucose, 0.5 mM L-glutamine, and 0 mM sodium
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pyruvate supplemented with 10% heat-inactivated FBS (56°C, 30 min), 100 U/ml penicillin G,
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100 µg/ml streptomycin and fungizone (250 µg/l).
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Experimental
Condition
6:
DMEM
with
0
mM
D-glucose,
0
mM
L-glutamine,
and
175
0 mM sodium pyruvate supplemented with 10% heat-inactivated FBS (56 °C, 30 min),
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100 U/ml penicillin G, 100 µg/ml streptomycin and fungizone (250 µg/l).
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After exposure, cells were trypisinized, counted and reseeded in 6-well plates at 800 cells per well. Cells were
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cultured under normal growth conditions (DMEM with 25.52 mM D-glucose, 4 mM L-glutamine, and 1 mM
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sodium pyruvate supplemented with 10% heat-inactivated FBS (56°C, 30 min), 100 U/ml penicillin G, 100
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µg/ml streptomycin and fungizone (250 µg/l) for 7 days to form colonies.
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Cell numbers were spectrophotometrically quantified with crystal violet as a DNA stain. Staining cell nuclei
184
of fixed cells with crystal violet allows for rapid, accurate and reproducible quantification of cell number in
185
cultures grown in culture plate [25,26]. After 7 days the medium was discarded and 600 µl of 1%
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glutaraldehyde (in FBS) was added to each well and incubated at room temperature for 15 min.
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Glutaraldehyde was discarded and 600 µl 0.1% crystal violet (in FBS) was added and left at room
188
temperature for 30 min. Crystal violet was discarded, micro-titer plates were immersed in running tap water
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for 10 min and left overnight to dry.
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Stained cells were solubilized by adding 1200 µl 0.2% Triton X-100 and incubated at room temperature for
192
30 min. The solution 100 µl (3 × ) was transferred to a clean 96-well micrometer plate and the absorbance was
193
read at 570 nm with an ELx800 Universal Microplate Reader from Bio-Tek Instruments Inc. (Vermont,
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United States of America).
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Quantification of fixated monolayer cells were spectrophotometrically determined by employing crystal
197
violet as a DNA stain. Cell survival was expressed as a percentage of the control which was set as 100%.
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Statistics
200
Data from the crystal violet assay were obtained from three independent biological replicates and each
201
biological replicate provided three technical replicates. The obtained data were statistically analyzed for
202
significance using a two-tailed Student’s t-test and two-way ANOVA and P-values < 0.05 were regarded as
203
statistically significant. Cell survival of the control was set to 100% and the means of the experimental
204
conditions were calculated relative to the control [27]. Means of the experimental conditions are represented
205
on the graph with error-bars referring to standard deviation.
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Results
208
209
Polarization-optical differential interference contrast and phase contrast (PlasDIC) light
210
microscopy
211
212
PlasDIC and phase contrast light microscopy were used to recognize and display morphological changes in
213
relation to other cells in order to gain insight on the effects that various exposures have on the cells. No major
214
morphological observations of increased apoptosis, reduction in cell density or cell shrinking were observed
215
in any of the experimental conditions after 2 h, 4 h or 6 h (Figs. 1, 2 and 3, respectively). However, 7 days
216
after reseeding the various experimental conditions differences in cell density were observed, as well as the
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formation of debris as a result of detached dead cells.
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HeLa cells that were exposed to 0 mM D-glucose, 0 mM L-glutamine, and 1 mM sodium pyruvate DMEM for
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2 h, 4h and 6h (Figs. 4D, 5D and 6D, respectively), 3 mM D-glucose, 0.5 mM L-glutamine, and 0 mM sodium
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pyruvate for 2 h, 4 h and 6 h (Figs. 4F, 5F and 6F, respectively) and DMEM with 0 mM D-glucose, 0 mM
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L-glutamine,
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displayed a decrease in cell density compared to their controls (Figs. 4A, 5A and 6A, respectively). Also, an
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increase in the formation of cellular debris in these samples, particularly pronounced in cells treated with 0
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mM D-glucose, 0 mM L-glutamine, and 0 mM sodium pyruvate for 6 h, was revealed (Fig. 6).
and 0 mM sodium pyruvate for 2 h, 4 h and 6 h (Figs. 4G, 5G and 6G, respectively) all
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These results suggest that immediate morphological changes after 2-6 h exposure are not apparent with
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PlasDIC light microscopy. However, the experimental conditions where D-glucose and L-glutamine levels
229
were below 6 mM and 1 mM for 2-6 h had a negative effect on colony formation and cell growth 7 days after
230
reseeding. Debris formation is also an indication of an increase in cell death with these experimental
231
conditions.
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We also investigated the effect of HeLa cells to withdrawal of glucose/glutamine and the glycolytic product
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pyruvate, which may serve as an alternate substrate for the TCA cycle. Pyruvate withdrawal resulted in only a
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minor ( ≈ 10%) loss of cell viability. This is consistent with previous reports [2].
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Fig. 1.
PlasDIC light microscopy (40 × ) showing influence of a 2 h exposure of HeLa cells to DMEM
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media containing various concentrations of D-glucose,
L-glutamine
or sodium pyruvate. (A) 0 h exposure, (B) 25.52 mM glucose, 4 mM glutamine, and 1 mM
240
pyruvate, (C) 6 mM glucose, 1 mM glutamine, and 1 mM pyruvate, (D) 3 mM glucose, 0.5 mM glutamine,
241
and 1 mM pyruvate, (E) 0 mM glucose, 0 mM glutamine, and 1 mM pyruvate, (F) 6 mM glucose, 1 mM
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glutamine, and 0 mM pyruvate, (G) 3 mM glucose, 0.5 mM glutamine, and 0 mM pyruvate, and (H) 0 mM
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glucose, 0 mM glutamine, and 0 mM pyruvate.
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Fig. 2.
PlasDIC light microscopy (40 × ) showing influence of a 4 h exposure of HeLa cells to DMEM
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media containing various concentrations of D-glucose,
L-glutamine
or sodium pyruvate. (A) 25.52 mM glucose, 4 mM glutamine, and 1 mM pyruvate, (B) 6 mM
249
glucose, 1 mM glutamine, and 1 mM pyruvate, (C) 3 mM glucose,
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0.5 mM glutamine, and 1 mM pyruvate, (D) 0 mM glucose, 0 mM glutamine, and 1 mM pyruvate, (E) 6 mM
251
glucose, 1 mM glutamine, and 0 mM pyruvate, (F) 3 mM glucose, 0.5 mM glutamine, and 0 mM pyruvate,
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and (G) 0 mM glucose, 0 mM glutamine, and 0 mM pyruvate.
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Fig. 3.
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PlasDIC light microscopy (40 × ) showing influence of a 6 h exposure of
HeLa cells to DMEM media containing various concentrations of D-glucose,
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L-glutamine
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glucose, 1 mM glutamine, and 1 mM pyruvate, (C) 3 mM glucose, 0.5 mM glutamine, and 1 mM pyruvate,
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(D) 0 mM glucose, 0 mM glutamine, and 1 mM pyruvate, (E) 6 mM glucose, 1 mM glutamine, and 0 mM
259
pyruvate, (F) 3 mM glucose, 0.5 mM glutamine, and 0 mM pyruvate, and (G) 0 mM glucose, 0 mM
260
glutamine, and 0 mM pyruvate.
or sodium pyruvate. (A) 25.52 mM glucose, 6 mM glutamine, and 1 mM pyruvate, (B) 6 mM
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Fig. 4. Phase contrast light microscopy (10 × ) showing influence of a 2 h exposure of HeLa cells to DMEM
263
media containing various concentrations of D-glucose, L-glutamine or sodium pyruvate.
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(A) 25.52 mM glucose, 4 mM glutamine, and 1 mM pyruvate, (B) 6 mM glucose, 1 mM glutamine, and
265
1 mM pyruvate, (C) 3 mM glucose, 0.5 mM glutamine, and 1 mM pyruvate, (D) 0 mM glucose, 0 mM
266
glutamine, and 1 mM pyruvate, (E) 6 mM glucose, 1 mM glutamine, and 0 mM pyruvate, (F) 3 mM glucose,
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0.5 mM glutamine, and 0 mM pyruvate, and (G) 0 mM glucose, 0 mM glutamine, and 0 mM pyruvate.
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Fig. 5. Phase contrast light microscopy (10 × ) showing influence of a 4 h exposure of HeLa cells to DMEM
271
media containing various concentrations of D-glucose, D-glutamine or sodium pyruvate.
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(A) 25.52 mM glucose, 4 mM glutamine, and 1 mM pyruvate, (B) 6 mM glucose, 1 mM glutamine, and
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1 mM pyruvate, (C) 0 mM glucose, 0.5 mM glutamine, and 1 mM pyruvate, (D) 0 mM glucose, 0 mM
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glutamine, and 1 mM pyruvate, (E) 6 mM glucose, 1 mM glutamine, and 0 mM spyruvate, (F) 3 mM glucose,
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0.5 mM glutamine, and 0 mM pyruvate, and (G) 0 mM glucose, 0 mM glutamine, and 0 mM pyruvate.
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Fig. 6. Phase contrast light microscopy (10 × ) showing influence of a 6 h exposure of HeLa cells to DMEM
278
media containing various concentrations of D-glucose, L-glutamine or sodium pyruvate.
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(A) 25.52 mM glucose, 4 mM glutamine, and 1 mM pyruvate, (B) 6 mM glucose, 1 mM glutamine, and
280
1 mM pyruvate, (C) 3 mM glucose, 0.5 mM glutamine, and 1 mM pyruvate, (D) 0 mM glucose, 0 mM
281
glutamine, and 1 mM pyruvate, (E) 6 mM glucose, 1 mM glutamine, and 0 mM pyruvate, (F) 3 mM glucose,
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0.5 mM glutamine, and 0 mM pyruvate, and (G) 0 mM glucose, 0 mM glutamine, and 0 mM pyruvate.
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Discussion
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All of the experimental conditions resulted in a reduction in the number of surviving cells when compared to
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the control. This indicates that reduction in D-glucose and L-glutamine levels in a dose-dependent manner
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from 6 mM and 1 mM and lower, respectively, have a negative effect on cell survival (Fig. 7A). Complete
297
removal of D-glucose and L-glutamine had the most significant adverse effect on cell survival (Fig. 7A) and
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an increase in the time of exposure to D-glucose and L-glutamine removal (E3 and E6) resulted in further
299
decreases in cell survival (Figure 7B).
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Fig. 7. Control: DMEM medium with 25.52 mM D-glucose, 4 mM L-glutamine, and 1 mM
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sodium pyruvate;
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E1: DMEM with 6 mM D-glucose, 1 mM L-glutamine, and 1 mM sodium pyruvate;
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E2:DMEM with 3 mM D-glucose, 0.5 mM L-glutamine, and 1 mM sodium
305
pyruvate;
306
E3: DMEM with 0 mM D-glucose, 0 mM L-glutamine, and 1 mM sodium pyruvate;
307
E4: DMEM with 6 mM D-glucose, 1 mM L-glutamine, and 0 mM sodium pyruvate;
308
E5: DMEM with 3 mM D-glucose, 0.5 mM L-glutamine, and 0 mM sodium
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310
pyruvate;
E6: DMEM with 0 mM D-glucose, 0 mM L-glutamine, and 0 mM sodium pyruvate.
311
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A) For 2 hours exposure; student t-test P-values less than 0.05 for E1 vs E3, E2 vs E6, E1
313
vs E6, E3 vs E6 and E4 vs E6.
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For 4 hours exposure; student t-test P-values less than 0.05 for E1 vs E3, E1 vs E5, E1 vs
315
E6, E2 vs E3, E2 vs E4, E2 vs E6, E3 vs E4, E3 vs E6, E4 vs E5, E4 vs E6, and E5 vs E6.
316
For 6 hours exposure; student t-test P-values less than 0.05 for E1 vs E3, E1 vs E6, E2 vs
317
E4, E2 vs E6, E3 vs E4, E3 vs E6, E4 vs E6, and E5 vs E6.
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319
B) For E3; student t-test P-values less than 0.05 for 2 hours exposure vs 6 hours exposure.
13
320
For E6; student t-test P-values less than 0.05 for 2 hours exposure vs 4 hours exposure and
321
2 hours exposure vs 6 hours exposure. Two-way ANOVA analysis (P-values less than 0.05)
322
suggested that an increase in the time of exposure to conditions E3 and E6 resulted in
323
decreased cell survival.
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The increased pro-oxidant status of cancer cells provides an opportunity for the selective killing of cancer
326
cells [15]. Reactive oxygen species (ROS) and an intracellular pro-oxidant status above a certain level
327
selectively activate pro-apoptotic SAPK pathways [28]. It is known that glucose deprivation is able to
328
increase oxidative stress as early as 1 h after the onset of glucose deprivation [29]. Glucose deprivation
329
results in increased formation of glutathione disulfide (GSSG), superoxide and hydrogen peroxide, resulting
330
in pro-oxidant status [28,29].
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Redox homeostasis abrogation in cancer cells is characterized by an intracellular pro-oxidant status as a result
333
of the cumulative production of ROS and the diminished capacity of antioxidant systems to balance the
334
excess production of ROS [28]. ROS activate pro-growth, pro-survival and proliferative signals though the
335
RAS-RAF-ERK1/2, PKB/AKT and NF-κB pathways [30,31].
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We argue that the initial 2-6 h glucose deprivation results in a pro-oxidant status in cancer cells. Since cancer
338
cells are more susceptible to oxidative stress due to their increased oxidative state [28,31], this initial increase
339
should result in oxidative damage to important biomolecules [32]. This initial damage possibly plays a causal
340
role in the subsequent abrogation of cell growth we observed and we think this provides a basis for future
341
research.
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Figure 7A further reveals that a reduction in D-glucose and L-glutamine levels has an adverse effect on HeLa
344
cell survival even after two hours of exposure. This demonstrates the possibility of killing cancer cells
345
through glucose deprivation in vitro. Recapitulating this putative therapeutic effect is difficult by only
346
cytotoxic means [33] due to, among others, the innocent bystander effect [15].
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When depriving the whole body of glucose and glutamine, safe concentrations are typically 2 mM and 1 mM,
349
respectively [19]. Extensive cancer cell killing is expected based on results from the present in vitro
350
experiment. Preliminary data show that carbohydrate restriction diets resulting in moderate ketosis can result
351
in stabilization or remission of cancer [14]. Such diet-induced suppression of glucose and insulin levels might
352
be more effective when combined with pharmacological treatments such as metformin, but this remains to be
353
proven [34].
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Wu et al. (2012) demonstrated that lactic acidosis resulted in resistance to cell death in glucose-deprived
356
cancer cells [35]. This suggests that disrupting lactic acidosis can be beneficial in conjunction with glucose-
357
deprivation of cancer cells. Dichloroacetic acid is an inhibitor of pyruvate dehydrogenase kinase 2 and
358
promotes the entry of pyruvate into oxidative phosphorylation, thus negatively affects fermentation and lactic
359
acid formation [36]. To enhance cancer cell killing, it seems to be prudent to combine glucose/glutamine
360
deprivation treatment, inhibitors of lactic acidosis such as DCA and other forms of chemoradiotherapy. Also,
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lower concentrations of cytotoxic agents would then be required, with lower adverse effects to surrounding
362
healthy tissue, meriting further investigation.
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Conclusion
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In vitro data reveal that glucose (and glutamine) deprivation to typical physiological concentrations result in
367
significant HeLa cancer cell killing after as little as 2 hours. Further in vivo studies are warranted to
368
investigate the possibility of combining glucose and glutamine deprivation with chemoradiotherapeutic
369
treatment of highly glycolytic cancers for enhanced cancer cell killing.
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Acknowledgements
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We acknowledge angel investor Dr Arnold van Dyk. TEMM International (Pty) Ltd, MCI (Pty) Ltd, and the
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University of Pretoria’s Department of Physiology (School of Medicine) co-funded the project. We thank
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Prof. Eugene Fine (Albert Einstein College of Medicine), Prof. Thomas Seyfried (Boston College), Prof.
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Douglas Spitz (University of Iowa), Dr. Rainer Klement (University Hospital of Würzburg), and Prof. Robert
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Gatenby (Moffitt Cancer Center) for proofreading the manuscript and for their valuable comments and
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suggestions.
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Competing interests
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The authors declare that they have no competing interests.
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Fig 1.
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containing various concentrations of D-glucose, L-glutamine
glutamine or sodium pyruvate. (A) 0 h exposure, (B) 25.52
464
mM glucose, 4 mM glutamine, and 1 mM pyruvate, (C) 6 mM glucose, 1 mM glutamine, and 1 mM
465
pyruvate, (D) 3 mM glucose, 0.5 mM glutamine, and 1 mM pyruvate, (E) 0 mM glucose, 0 mM glutamine,
glutamin
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and 1 mM pyruvate, (F) 6 mM glucose, 1 mM glutamine, and 0 mM pyruvate, (G) 3 mM glucose, 0.5 mM
467
glutamine, and 0 mM pyruvate, and (H) 0 mM glucose, 0 mM glutamine, and 0 mM pyruvate.
PlasDIC light microscopy (40 × ) showing influence of a 2 h exposure of HeLa cells to DMEM media
20
468
469
Fig. 2.
470
media containing various concentrations of D-glucose, L-glutamine
glutamine or sodium pyruvate. (A) 25.52 mM
471
glucose, 4 mM glutamine, and 1 mM pyruvate, (B) 6 mM glucose, 1 mM glutamine, and 1 mM pyruvate, (C)
472
3 mM glucose, 0.5 mM glutamine, and 1 mM pyruvate, (D) 0 mM glucose, 0 mM glutamine, and 1 mM
473
pyruvate, (E) 6 mM glucose, 1 mM glutamine, and 0 mM pyruvate, (F) 3 mM glucose,
474
0.5 mM glutamine, and 0 mM pyruvate, and (G) 0 mM
mM glucose, 0 mM glutamine, aand 0 mM pyruvate.
PlasDIC light microscopy (40 × ) showing influence of a 4 h exposure of HeLa cells to DMEM
21
475
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Fig. 3.
PlasDIC light microscopy (40 × ) showing influence of a 6 h exposure of
477
HeLa cells to DMEM media containing various concentrations of D-glucose, L-glutamine
glutamine or sodium pyruvate.
478
(A) 25.52 mM glucose, 6 mM glutamine, and 1 mM pyruvate, (B) 6 mM glucose, 1 mM glutamine, and
479
1 mM pyruvate, (C) 3 mM glucose, 0.5 mM glutamine, and 1 mM pyruvate, (D) 0 mM glucose, 0 mM
480
glutamine, and 1 mM pyruvate, (E) 6 mM glucose, 1 mM glutamine, and 0 mM pyruvate, (F) 3 mM glucose,
481
0.5 mM glutamine, and 0 mM pyruvate, and (G) 0 mM
mM glucose, 0 mM glutamine, and 0 mM pyruvate.
22
482
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Fig. 4. Phase contrast light microscopy (10 × ) showing influence of a 2 h exposure of HeLa cells to DMEM
484
media containing various concentrations of D-glucose, L-glutamine
glutamine or sodium pyruvate.
485
(A) 25.52 mM glucose, 4 mM glutamine, and 1 mM pyruvate, (B) 6 mM glucose, 1 mM glutamine, and
486
1 mM pyruvate, (C) 3 mM glucose, 0.5 mM glutamine, and 1 mM
M pyruvate, (D) 0 mM glucose,
gl
0 mM
487
glutamine, and 1 mM pyruvate, (E) 6 mM glucose, 1 mM glutamine, and 0 mM pyruvate, (F) 3 mM glucose,
488
0.5 mM glutamine, and 0 mM pyruvate, and (G) 0 mM
mM glucose, 0 mM glutamine, and 0 mM pyruvate.
489
23
490
491
Fig. 5. Phase contrast light microscopy (10 × ) showing influence of a 4 h exposure of HeLa cells to DMEM
492
media containing various concentrations of D-glucose, D-glutamine
glutamine or sodium pyruvate.
493
(A) 25.52 mM glucose, 4 mM glutamine, and 1 mM pyruvate, (B) 6 mM glucose, 1 mM glutamine, and
494
1 mM pyruvate, (C) 0 mM glucose, 0.5 mM glutamine, and 1 mM pyruvate, (D) 0 mM glucose, 0 mM
495
glutamine, and 1 mM pyruvate, (E) 6 mM glucose, 1 mM glutamine, and 0 mM spyruvate, (F) 3 mM glucose,
496
0.5 mM glutamine, and 0 mM pyruvate, and (G) 0 mM
mM glucose, 0 mM glutamine, and 0 mM pyruvate.
24
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Fig. 6. Phase contrast light microscopy (10 × ) showing influence of a 6 h exposure of HeLa cells to DMEM
499
media containing various concentrations of D-glucose, L-glutamine
glutamine or sodium pyruvate.
500
(A) 25.52 mM
M glucose, 4 mM glutamine, and 1 mM pyruvate, (B) 6 mM glucose, 1 mM glutamine, and
501
1 mM pyruvate, (C) 3 mM glucose, 0.5 mM glutamine, and 1 mM pyruvate, (D) 0 mM glucose, 0 mM
502
glutamine, and 1 mM pyruvate, (E) 6 mM glucose, 1 mM glutamine, and 0 mM pyruvate, (F) 3 mM glucose,
503
0.5 mM glutamine, and 0 mM pyruvate, and (G) 0 mM glucose, 0 mM glutamine, and 0 mM pyruvate.
504
505
25
506
507
Fig. 7. Control: DMEM medium with 25.52 mM D-glucose,
glucose, 4 mM L-glutamine, and 1 mM
508
sodium pyruvate;
509
E1: DMEM with 6 mM D-glucose, 1 mM L-glutamine,
glutamine, and 1 mM sodium pyruvate;
510
E2: DMEM with 3 mM D-glucose, 0.5 mM L-glutamine,
glutamine, and 1 mM sodium
511
pyruvate;
512
E3: DMEM with 0 mM D-glucose, 0 mM L-glutamine,
glutamine, and 1 mM sodium pyruvate;
513
E4: DMEM with 6 mM D-glucose, 1 mM L-glutamine,
glutamine, and 0 mM sodium pyruvate;
514
E5: DMEM with 3 mM D-glucose, 0.5 mM L-glutamine,
glutamine, and 0 mM sodium
515
pyruvate;
26
516
E6: DMEM with 0 mM D-glucose, 0 mM L-glutamine, and 0 mM sodium pyruvate.
517
518
A) For 2 hours exposure; student t-test P-values less than 0.05 for E1 vs E3, E2 vs E6, E1
519
vs E6, E3 vs E6 and E4 vs E6.
520
For 4 hours exposure; student t-test P-values less than 0.05 for E1 vs E3, E1 vs E5, E1 vs
521
E6, E2 vs E3, E2 vs E4, E2 vs E6, E3 vs E4, E3 vs E6, E4 vs E5, E4 vs E6, and E5 vs E6.
522
For 6 hours exposure; student t-test P-values less than 0.05 for E1 vs E3, E1 vs E6, E2 vs
523
E4, E2 vs E6, E3 vs E4, E3 vs E6, E4 vs E6, and E5 vs E6.
524
525
B) For E3; student t-test P-values less than 0.05 for 2 hours exposure vs 6 hours exposure.
526
For E6; student t-test P-values less than 0.05 for 2 hours exposure vs 4 hours exposure and
527
2 hours exposure vs 6 hours exposure. Two-way ANOVA analysis (P-values less than 0.05)
528
suggested that an increase in the time of exposure to conditions E3 and E6 resulted in
529
decreased cell survival. * indicates a t-test P-value of < 0.05.
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531
27