archives of oral biology 58 (2013) 563–574

Available online at www.sciencedirect.com

journal homepage: http://www.elsevier.com/locate/aob

Review

Ascorbic acid and its pro-oxidant activity as a therapy for

tumours of oral cavity – A systematic review

Manisha Chandini Putchala *, Pratibha Ramani, Herald J. Sherlin, Priya Premkumar,

Anuja Natesan
Department of Oral and Maxillofacial Pathology, Saveetha Dental College, Chennai, India

article info abstract

Article history: Background: Ascorbic acid or Vitamin C is a potent dietary antioxidant with a double faced
Accepted 30 January 2013 character, in that it exhibits a pro-oxidant activity arising from its routine antioxidant
property that generates reactive free radicals, which induce cytotoxic effects at pharmaco-
Keywords: logic concentrations. A systematic review of this effect of ascorbic acid in the oral tumours
Ascorbic acid and normal oral tissues would clearly elucidate the merits or demerits of employing vitamin
Pro-oxidant activity C in treating the same.
Treatment Objective: The aim of our systematic review is to critically review the studies reported in
Oral neoplasms literature that have studied the pro-oxidant activity of ascorbic acid as a therapeutic option
for treatment of oral neoplasms and its effects on normal oral cells.
Methods: Articles were searched in PUBMED, MEDLINE using appropriate key words like
‘‘ascorbic acid’’, ‘‘pro-oxidant activity’’, ‘‘treatment’’, ‘‘oral neoplasms’’. Hand search of
Journals was also performed. Articles were reviewed and analysed.
Results: The search strategy included 17 potentially relevant articles for review of which, 12
were in vitro studies; 3 were in vivo animal studies; 1 was in vivo human study and 1 was ex
vivo human study. The optimum concentration of ascorbic acid used to produce potential
pro-oxidant associated cytotoxic effects was found to be 3–5 mM in vitro, 0.88–5 mM in vivo
animals, 0.5–2 mM ex vivo in humans, and the corresponding effects are induction of
apoptosis (caspase activation), necrosis, free radical formation, H2O2 generation, and
DNA fragmentation. In contrast, the same pro-oxidant concentrations had no effect on
the normal cells.
Conclusion: The results of our systematic review show that the pro-oxidant activity of
pharmacologic ascorbic acid is a part of its dose-dependent bimodal activity and is a result
of the proposed Fenton mechanism. In vitro, animal and ex vivo studies of pharmacologic
ascorbic acid (AA) have yielded meritorious results proving vitamin C as an effective
cytotoxic agent against oral neoplastic cells with potentially no harming effects on normal
cells. However, a shortage of clinical trials and in vivo human studies pertaining to
evaluation of anti-tumour activity of vitamin C in tumours of oral cavity remains a lacuna
in concluding ascorbic acid as a beneficial therapeutic option in treatment of oral neo-
plasms.
# 2013 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: +91 9790957986.

E-mail addresses: manishachandini@gmail.com, mani7chandini@gmail.com (M.C. Putchala).
0003–9969/$ – see front matter # 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.archoralbio.2013.01.016
564 archives of oral biology 58 (2013) 563–574

Contents

1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
1.1. Antioxidant ascorbic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
1.2. Pro-oxidant ascorbic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
1.3. Review of literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564
2. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
2.1. Search strategy for identification of studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
2.2. Search methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
2.3. Selection criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
2.4. Data extraction and analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
2.5. Outcomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566
3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566
3.1. Included studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566
4.1. Outcomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
5.1. Implications for practice and research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
5.2. Limitations of the review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572

1.2. Pro-oxidant ascorbic acid

1. Background
Ascorbic acid or Vitamin C is a water soluble vitamin that has
gained importance over years by distinguishing itself from the
rest by exhibiting a spectrum of biological functional proper-
ties. Vitamin C is a dietary antioxidant, classified under the
group of primary or natural antioxidants.1 While lower
vertebrates having the ability to self-synthesize the vitamin,
apes and humans cannot synthesize Ascorbic acid due to the
lack of an enzyme gulonolactone oxidase that catalyzes the
terminal step in the synthesis of L-ascorbic acid from the
substrates, D-glucose or D-galactose.2
The chemical name of ascorbic acid is 2-oxo-L-threo-
hexono-1,4-lactone-2,3-enediol and it exists in two major
dietary forms: L-ascorbic acid, a reduced form and dehy-
droascorbic acid (DHA), an oxidised form. Ascorbic acid is
readily absorbed by active transport in the intestine. A body
pool of 1.2–2.0 g of ascorbic acid is maintained by intake of
75 mg/day. Its average half life in adult human is about 10–20
days. The main route of elimination is through urine and the
major metabolites are DHA, 2,3-ketogulonic acid and oxalic
acid.2
1.1. Antioxidant ascorbic acid
Ascorbic acid is a potent antioxidant or reducing agent that
is capable of scavenging free radicals of reactive oxygen and
nitrogen species (ROS and RNS) that have potential to
damage nucleic acids and promote carcinogenesis.3,4 The
physiological functions are largely dependent on its oxido-
reductive properties. The ascorbate reacts with ROS,
quenches them and gets converted into poorly reactive
semi-hydroascorbate radical. Therefore ascorbate efficiently
decreases in vivo damage to proto-oncogenes and tumour
suppressor genes, thereby reducing the risk of cancer by
suppressing the oxidative stress induced by reactive free
radicals.3
Ascorbic acid presents a double faced character in that it
exhibits a pro-oxidant activity arising from its routine antioxi-
dant property. Apart from reducing oxidising sources like H2O2,
ascorbate also reduces metal ions like Fe3+ and Cu3+, the process
during which free radicals are generated. This reaction in which
ascorbate generates highly reactive free radicals in presence of
transition metal ions is called Fenton reaction.2,5
2Fe3þ þ Ascorbate ! 2Fe2þ þ DHA
2Fe2þ þ 2H2 O2 ! 2Fe3þ þ 2OH þ 2OH
These hydroxyl radicals are reported to interact with DNA
inducing its damage by causing breaks in phosphodiester
backbone and modification of DNA bases.3 This property of
pro-oxidant activity inducing cytotoxicity has been employed
in many studies in the prevention and treatment of cancers
and is proposed to be dose-dependent. On the other hand,
there are existing controversies questioning the activity of
ascorbic acid whether, it is beneficial in the treatment of
tumours or antagonizes the oxidative stress induced by
traditional therapeutics by providing antioxidant protection
to tumour cells.
1.3. Review of literature
Mamede et al.4 and Naidu2 stated that the ROS generated in
response to high concentration of vitamin C can catalyze lipid
peroxidation and are cytotoxic to cancer cells; the concept that
was also advocated by Verrax and Calderon.6 Koch and
Baiglow7 also reported this activity results in damage to cell
membranes and DNA, unless neutralized by catalase. Benade
et al.8 suggested that tumour cells are often catalase deficient,
thereby being more sensitive to H2O2 than normal cells. Augus
et al.9 and Langemann et al.10 reported accumulation of
vitamin C at high concentrations in solid tumours than in
normal surrounding cells.
 archives of oral biology 58 (2013) 563–574
Rozanova et al.11 advocated the use of vitamin C in
conjugation with medical herbs in treatment of carcinoma
cell lines in which stimulation of apoptosis and disruption of
cell cycle has shown to occur. Heaney et al.12 employed pre-
treatment with DHA in treating few malignant cell lines.
Casciari et al.13 not only reported the cytotoxic effect of
ascorbate in in vitro hollow fibre colon tumour but also studied
the effects of ascorbic acid on doxorubicin therapy. The study
concluded that low doses of ascorbic acid offered antioxidant
protection to tumour cells while high doses increased cell
killing. Similarly, a study done by Raloff14 suggested that
vitamin C may provide antioxidant protection to tumour cells
from routine chemotherapeutics.
Chen et al.15 published an in vivo study in rats where pro-
oxidant activity of vitamin C was achieved at human
pharmacologic concentrations of the vitamin. Yeom et al.16
tested the carcinostatic effects of ascorbic acid in mice with
sarcoma cells in which he reported a 20% increase in the
survival rate in high dose group. Riordan et al.17 reported that
oral supplementation of vitamin C is unlikely to produce
cytotoxic concentration levels while higher intravenous doses
had been proven effective. Varga and Airoldi18 and Tsao et al.19
also reported increased host survival time and inhibition of
tumour growth following ascorbate administration in rats.
Hoffer et al.20 reported the anti-tumour activity of vitamin C
in advanced cases of hematologic malignancy with intravenous
infusions thrice a week. Cameron and Campbell21 and Murata
et al.22 reported in a clinical trial that the terminal cancer
patients when given IV vitamin C at dose of 10 g/day showed
longer survival times than the controls. However, a clinical trial
carried out at Mayo clinic showed no significant benefits
between vitamin C administered and placebo groups.23
Pertaining to the neoplasms of oral cavity, majority of the
studies that have evaluated the proxidant activity of vitamin C
are in vitro studies. A study done by Chu et al.24 demonstrated
increased cytotoxic activity in human oral squamous cell
carcinoma cell lines when treated with vitamin C. Kishino
et al.25 reported profound anti-tumour potential of an ascorbic
acid derivative, sodium 5,6-benzylidene-L-ascorbate in human
oral squamous cell carcinoma cell lines. Not only the oral
cancers, but vitamin C was also reported to significantly
suppress the incidence of potentially malignant lesions like
oral leukoplakia in induced animal models as demonstrated
by Harada et al.26
With various clinical and scientific evidences of the
cytotoxic effect of ascorbic acid through its pro-oxidant
activity, we strongly opine a systematic review of this effect
of ascorbic acid in the oral neoplasms and normal oral tissues
would clearly elucidate the merits or demerits of employing
vitamin C in treating the same.
2. Methods
2.1. Search strategy for identification of studies
The search strategy was in accordance with the Cochrane
guidelines for systematic reviews. Articles were searched and
selected using PUBMED, MEDLINE till the year 2012. Addition-
ally Google Scholar was also used to obtain the relevant
565
articles of our interest. Due to scarcity of studies in oral tissues
we wished to exhaust all the possible articles; accordingly no
timeline was included in the search (search results beginning
from the year 1977). The article search included only those
published in the English literature. The title of the articles and
abstracts were reviewed.
2.2. Search methodology
The search methodology through PUBMED was done using the
following keywords:
Search (ascorbic acid) OR (vitamin C) OR (sodium ascorbate)
OR (dehydroascorbate) AND (pro-oxidant) OR (prooxidant
activity) OR (vitamin C prooxidant) OR (ascorbic acid
prooxidant) OR (prooxidant effects) OR (prooxidant prop-
erty) AND (chemotherapy) OR (treatment) AND (oral
diseases) OR (oral pathologies) OR (oral lesions) OR (oral
neoplasms) OR (oral proliferations) OR (oral enlargements)
OR (oral hyperplasias) OR (oral hypertrophies).
In addition, an internet search was also done through
Google Scholar using the key words ‘‘vitamin C’’ and ‘‘pro-
oxidant activity’’ and ‘‘oral lesions’’.25,26,29,33,35,42,47,49 Journals
evaluating the pro-oxidant activity of vitamin C in cancers
were also used from cross references.36,41 The process is
summarised in Fig. 1.
2.3. Selection criteria
Inclusion criteria:
 Studies that evaluated pharmacologic dose associated pro-
oxidant activity of vitamin C or ascorbic acid in oral
neoplasms.
 Studies that analysed pharmacologic dose effects of vitamin
C in healthy oral cells and tissues.
 Studies that evaluated pro-oxidant effects of vitamin C in
cell lines that have implications in oral neoplasms.
 Studies that measured pro-oxidant concentrations of
ascorbic acid blood, serum, extracellular fluid (plasma).
 Studies that included vitamin C as positive control in
measuring the cytotoxic effects of other antioxidants in oral
cell lines.
 Studies that included ascorbic acid as cytotoxicity enhancing
agent in combination with other materials in oral cell lines.
Exclusion criteria:
 Studies that have studied traditional antioxidant property of
vitamin C or ascorbic acid in treatment of oral diseases.
 Studies that evaluated antitumour potential of vitamin C in
general neoplasms other than oral pathologies.
 Review articles.
2.4. Data extraction and analysis
Once the potentially relevant articles for systematic review
were obtained, data extracted from each article was tabulated
and was later cross checked.
566 archives of oral biology 58 (2013) 563–574
2.5. Outcomes
The outcomes assessed in this review were the optimum
concentration or level of ascorbic acid to exert a pro-oxidant
effect in vivo and in vitro, effects of pharmacologic dose
supplementation of vitamin C on normal oral and tumour cell
lines; duration of ascorbic acid (AA) administration and
potential benefits of vitamin C employment in treatment of
various oral neoplasms.
3. Results
The selection and exclusion criteria of the reviewed studies
are summarised in Fig. 1. The search strategy identified
seventeen studies that evaluated pro-oxidant activity of
vitamin C in various oral cell lines. The description of the
included studies is shown in Table 1.
3.1. Included studies
The search strategy included 17 potentially relevant articles
for review of which, 12 were in vitro studies; 3 were in vivo
animal studies; 1 was in vivo human study and 1 was ex vivo
human study. The parameters measured were many and
differed from study to study in evaluating the pro-oxidant
[(Fig._1)TD$IG]
Fig. 1 – Search flowchart.
effects of vitamin C. Most of the studies measured the
percentages of apoptotic cells, viable cells, positive cells of
caspase activation, positive cells of free radical and H2O2
generation, DNA fragmentation; levels of DNA adducts, and
tumour growth. EC50 or CC50 of ascorbic acid in different
neoplastic and normal oral cell lines was also evaluated.
4. Discussion
Exertion of pro-oxidant effect by high doses of VC was
reported to occur against various cancer cell lines through
Fenton reaction in presence of transition metal ions.3,27 The
normal cells on the other hand, having redundant mecha-
nisms for H2O2 disposal and/or repair of H2O2 damage remain
resistant to the pro-oxidant concentrations of AA unlike the
susceptible cancer cells which lack these mechanisms (e.g.,
catalase deficiency, mutated DNA repair and tumour suppres-
sor genes) and may have series of mutations that signal cell
death in context of H2O2 formed by pharmacologic ascor-
bate.28
The present systematic review discusses the various
studies that have tested, assessed the mechanism of action
of pharmacologic ascorbate against different abnormal oral
cells and tissues, of which majority are studies done in vitro.
Bonilla-Porras et al.29 have reported induction of apoptosis in
 archives of oral biology 58 (2013) 563–574 567

Table 1 – Description of included studies in the chronological order of publication.

S. Author & Type of Agent Coadmin Cell type/ Conc. of Evaluated Parameters Results
No Year of Study tested -istered Tissue type Asa/Vit
. Publication agents C
1. Bonilla- In-vitro Vitamin C Vitamin Case: 1mM % positive apoptotic nuclei At 5mM VC,
Porras et al Case- (VC). K3 Leukemia 5mM Lymphocytes = 2±1%
[29]. Control (VK3) cells- 10mM K562 = 100%
2011 study VC in 1) Jurkat clone 20mM Jurkat = 5±2%
combination E6-1 At 20mM VC
. 2) K562 Lymphocytes = 3±1%
Control: K562 = 100%
Lymphocytes Jurkat = 77±2%
% positive Jurkat cells
[TD$INLE] with: At 10mM VC:
• O2- generation 13%
• H2O2 generation 28±2%
• Mitochondrial 73%
membrane damage
2. Deubzer et In-vitro Ascorbate ------ Neuroblastoma 0.6mM Percentage of viable cells: At 1.3mM Asc,
al [33]. observa- (Asc) cell lines- 1.3mM • Kelly – 3.5x104 ≈ 70±3%
2010 tional 1) Kelly cells in 2.5mM 10x104 ≈ 62±2%
study concentrations 5.0mM 25x104 ≈ 90±2%
of 3.5x104, At 5mM Asc,
10x104, 3.5x104 ≈ 3±1%
25x104. 10x104 ≈ 4±1%
2) SK-N-SH 25x104 ≈ 70±5%
cells in • SK-N-SH – At 1.3mM Asc,
concentrations 2.5x104 ≈ 21±2%
of 2.5x104, 9x104 ≈ 61±5%
9x104, 14x104. 14x104 ≈ 75±2%
At 5mM Asc,
2.5x104≈ 10±1%
9x104 ≈ 12±1%
14x104≈ 50±3%
3. Chu et al In-vitro Three herbal Sodium Human Oral 0-4mM • DNA HL-60: intranucleosomal
[24]. observa- extracts of ascorbate squamous cell fragmentation(qualit DNA fragmentation positive.
2009 tional Drynaria (Sod.asc) carcinoma cell ative) HSC-2: Positive smear pattern
study baronii lines: HSC-2, of DNA fragmentation.
(DB), Sod.asc NA.
[TD$INLE] Angelica in Human • Caspase 3,8,9 HL-60:caspase activation
sinensis combinati promyelocytic activation evident
(AS), on. leukemia cell (qualitative) HSC-2: Feeble activation of
Cornus line: HL-60 caspases.
officinalis
(CO) • Combination effect Combination of DB or CO but
of cytotoxicity. not AS, with 1mM sod.asc
enhanced cytotoxicity.
4. Belin et al In-vitro Ascorbic ------ Raji cells 0-3mM Cell density estimation 0mM AA 70
[36]. observat acid (AA) (units- not mentioned), at 0.6mM AA 28
2009 ional 70 hrs 2mM AA 9
study 3mM AA 1

Cell density estimation 0.6mM/R 65

with Reversal effects of 2mM/R 20
Asa withdrawal, at 70 hrs. 3mm/R 5

% cells blocked at: With 0mM & 3mM AA

respectively:
• G0/G1 phase 41% ; 50%
• S phase 42% ; 50%
• G2/M phase 15% ; 0%
• Cell death 0% ; 15%

% dead cells at 48hrs & 0.6mM 1% & 40%

96hrs respectively. 2mM 20% & 100%
[TD$INLE] 3mM 100%

% cells after 24 hours: 0.6mM 2mM 3mM

• Viable 64% 30% 15%
• Apoptotic 17% 20% 15%
16% 41% 65%
• Necrotic
5. Kishino et al In-vitro Sodium 5,6- ------ Human oral 0-8mM CC50 of SBA. CC50 SBA (mean):
[25]. observat Benylidene- squamous cell Normal oral cells = 4.8mM
2008 ional L-ascorbate carcinoma cell HSC-2,3,4 = 2.33mM
study (SBA) lines: HSC-2, Leukemia cell lines =
HSC-3, HSC- 1.37mM
4.
Tumor specificity index TS = 2.
Human (TS).
568 archives of oral biology 58 (2013) 563–574

myelogenous Caspase activation Little or no activation of

leukemia cell (qualitative) caspases 3,8,9.
lines: HL-60,
ML-1, KG-1. DNA fragmentation Smear pattern of DNA
(qualitative) fragmentation in HSC-2 and
Human normal HSC-4 on prolnged treatment
oral cells: (24h).
HGF, HPC,
HPLF. Ultra-structural changes in HSC-2 cells: Mitochondrial
cells swelling, enlargement of ER
at 4mM SBA. Rupture of cell
membrane, discharge of
damaged cell organelles at
[TD$INLE] 8mM SBA.
HSC-4 cells: Slight and
aggrevated mitochondrial
damage at 2mM and
4mM,8mM SBA respectively.
6. Chen et al In-vitro Ascorbate ------ Normal cell 0-20mM EC50 of Asc (mM) Normal cell lines: >20mM.
[37]. observat (Asc). lines: B16: 8mM.
2008 ional Monocyte JLP119: 1mM.
study Lymphocyte
Fibroblast: Cytotoxicity in normal cell Not evident.
WI38, lines.
CCD34SK
% viable cells in fibroblast At 1mM Asc- 77%
Tumour cell cell lines At 10mM Asc- 75%
lines:
Melanoma
(B16)
Lymphoma
(JLP119)
7. Carosio et al In-vitro Sod.asc ------ Neuroblastoma 0.5mM- EC50 (mM) after 24 hrs. <2mM for all cell lines.
[35]. observat cell lines: 3mM.
2007 ional HTLA-230 % Apoptotic cells after HTLA-230 = 87±6%
study. IMR-32 1day incubation with 3mm IMR-32 = 60±4%
LAN-5 sod.asc LAN-5 = 60±4%
GI-LI-N GI-LI-N = 93±5%
SH-SY5Y SH-SY5Y = 90±5%
% positive caspase
[TD$INLE] activated cells of: (mM) 0 1 1.6 2 3
HTLA-230 (%) 17 25 60 75 95
SH-SY5Y 13 30 65 75 85
after 1day incubation with
sod.asc (mM)
8. Chen et al In-vivo Ascorbate ------ Blood. 0.7mM - Asa(acid) conc. in IV= >8mM peak in 4 min
[15]. animal Extracellular 1.4mM plasma(mM for IV, IP; μM IP≈ 3mM peak in 15 min
2007 observat fluid. by three for gavage). Gavage = 50μM - <100μM
ional routes: steady over 10 - 120 min.
study. IV(n=7)
Animal IP (n=5) Asa conc. in ECF (mM). IV≈ 8mM peak in 30 min
model: Gavage IP≈ 3mM peak in 60 min
rat (n=5) gavage≈ 125-130μM peak
in 60 min.
Correlation between Asa High positive correlation.
conc. of plasma & ECF.

Asc*- conc. in ECF (nM). IV= 250nM peak in 30 min

IP≈150nM peak in 60 min
Gavage = <50nM

Asc*- conc. in blood (nM). IV & IP≈ 30-40nM

Gavage – undetectable.

H2O2 conc. in ECF (μM). IV≈ 20μM peak at 30 min

IP≈ not >5μM.
[TD$INLE] Gavage = no change.
Correlation between H2O2
conc. in ECF and Asc conc.
in blood or in ECF. Positive linear correlation
9. S. H. Bhat et Ex-vivo AA Cupric Human 0.05- DNA breakage through 0.05mM AA 15μ metres
al [42]. human chloride lymphocytes 0.5mM comet tail length (μ 0.1mM AA 50μ metres
2006 observat AA in (Cu(II))- metres). 0.4mM AA 95μ metres
ional combination 20μM. 0.5mM AA 120μ metres
study . Neocupro
ine (NC)- Effect of neurocuproine 0.1mM NC 57μ metres
100- on Asa induced DNA 0.2mM NC 30μ metres
400μM. breakage, through comet 0.4mM NC 17μ metres
tail length.
 archives of oral biology 58 (2013) 563–574 569

10 Chen et al In-vitro AA. H2 O 2 Human 0.1-5mM EC50 of AA (mM) JLP-119: 1mM

. [41]. and ex- Burkitt’s Normal cell lines: >20mM
2005 vivo Dehydroasc lymphoma
observat orbic acid (JLP-119). % cell death in: (mM) 0.5 1 1.5 2
ional (DHA). • JLP-119 (%) 60 80 90 91
study Normal cell • Lymphocyte 19 19 20 22
lines: • Monocyte 7 7 11 10
Fibroblast
(CCD34SK) % cell death in JLP-119
Lymphocyte with: (mM) 0.5 1 1.5 2
Monocyte • AA (%) 60 80 90 95
• DHA 5 5 7 10
Human RBC
[TD$INLE] H2O2 conc. (μM)after 1hr 0.2mM 30μM
incubation with AA (mM). 0.5mM 50μM
1mM 100μM
2mM 12μM
% cell death with added
H2O2 (μM) in: (μM) 50 100 250
• JLP-119 (%) 50 65 70
10 15 20
• Lymphocyte
10 10 19
• Monocyte

Asc*- formation (nM) by:

(mM) 2 5 10
• AA(mM)
(nM) 55 75 100
• Medium 0 7 10
• Blood
H2O2 formation (μM) on Medium: 160μM at peak
addition of AA to medium Blood: 0-1μM
and blood.

11 Kinoshita et In-vitro Dental Sodium Human oral 0.25mM Cytotoxicity of Sod.asc 0.25mM sod.asc
. al [43]. observa- metals: Cu, ascorbate squamous cell combination with metals in combination significant
2002 tional Pd-alloy, carcinoma cell 0.5-2mM HSC-2 cells. reduction in growth inhibitory
study Ag, Au. lines: HSC-2, effect of Pd-alloy.
HSC-3. 15mM in 0.5-2mM Sod.asc
Human 0.1mM combination enhanced
submandibular Tris-HCL growth inhibitory action of Cu
gland and Pd-alloy.
carcinoma
[TD$INLE]
(HSG). Relative viable HSC-2 45%-50% of the control, from
Human cells expressed as % of the combinations with Cu or Pd
promyelocytic control (cell line without alloy
leukemia cell addition of combination)
line (HL-60)
Human normal Radical intensity of Cu- enhanced radical intensity
oral cell lines: ascorbate analysed by ESR of Asc followed by rapid its
HGF, HPC, spectroscopy. decline.
HPLF. Pd-alloy- enhanced ascorbate
radical intensity for longer
period (30 min).
Ag, Au- did not affect radical
intensity of sod.asc.
12 Sakagami et In-vitro Sodium L- ------ Human oral 1-5mM CC50 of sodium HSC-2: 2.3mM
. al [44]. observat ascorbate tumour cell ascorbate. HSG: 5.63mM
2001 ional lines: HSC-2, HL-60 cells: 0.23mM
study HSG.
Human H2O2 production 1mM of Asc at pH 10: Rapid
myeloblastic reflected through production, but significantly
leukemia cell chemiluminiscence assay at lower amount of H2O2 (105)
line: Ml-1 different pH variations. within 30 min. At pH 6: 103
Human
promyelocytic Radical generation At pH 7.4 = 0.31, pH 9= 2.77
leukemia cell through ESR spectra(spike
line: HL-60 units).

[TD$INLE] DNA fragmentation and Weakly potent.

caspase activation.
13 Vojdani et al In-vivo Ascorbic ------ Human Gr I: Intracellular levels of Gr I = 7.0±2.26
. [45]. human acid. peripheral baseline ascorbic acid (μg/5x106 Gr II= 10.1±1.2
2000 observat blood Gr II: cells) at 15th day. Gr III= 10.7±2.8
-ional leukocytes. 2.83mM Gr IV= 9.5±1.9
study. Gr III:
4 groups 5.67mM Level of 8- Gr I = 7.6±5.9
(Gr) Gr IV: hydroxyguanosine bases- Gr II = 4.6±1.5
with 28.38mM DNA adducts (/106 guanine Gr III = 7.0±4.6
varying Per day bases) at 15th day. Gr IV = 7.4±6.7
doses. for
2weeks NK cell cytotoxic Gr I = 34.4±26.3
with 1 activity (lysis units LU) at Gr II = 52.4±24.4
570 archives of oral biology 58 (2013) 563–574

week of 15th day Gr III = 77.6±36.4

washout. Gr IV = 35.0±24.9

% leukocyte apoptosis at Gr I = 8.2±4.6

15th day Gr II = 5.1±2.5
Gr III = 3.9±2.3
Gr IV = 5.5±2.8
14 E. E Kelley In-vitro L-ascorbic Photofrin Human oral 0.1mM Lipid free radical intensity. Lipid radical intensity: 8±2
. et al [46]. observa- acid in (porfimer squamous cell arbitrary units as compared to
1997 tional combination sodium) - carcinoma cell 1.6±0.2 units without pro-
study . 15μg/ml. line: SCC-25 oxidant mixture.
Ferrous
[TD$INLE] sulphate - Percentage of surviving 3±2% as compared to 24±4%
20μM cell proportion without pro-oxidant mixture,
Visible at given time.
light
15 Noto et al In-vitro Sodium Vitamin Human 0.001- Percentage of growth At 5mM of vit C alone =
. [47]. observat ascorbate K3 Oral 10mM inhibition of cells. 50%.
1989 ional (vitamin C) epidermoid Vit C
study alone. (KB) cell lines 100:1 In combination,
Vitamin C concentra 0.5mM Vit C + 0.005mM
in tion of Vit K3 = 50%.
combination vitC:vitk3
. (combinat 1mM Vit C + 0.01mM Vit
ion). K3 = 100%
16 Harada et al In-vivo Case Diethyl- Tongue 5.67mM Percentage incidence of Case & Control
. [26]. case (n=30): nitrosami Hard palate leukoplakia. Tongue = 75% & 93%
1987 control Vitamin C. ne (DEN) Soft palate Hardpalate = 50% & 90%
animal Control Cigarette Buccal mucosa Softpalate = 32% & 72%.
study. (n=30): no smoke. Pharynx Buccal mucosa=7% & 17%
model - vitamin C Pharynx = 43% & 67%
Hamster
17 J. A. In-vivo Ascorbic 20- Mesenchymal Groups: Tumour growth (cm2) Mean Tumor size from 4th-
. Migliozzi observa- acid. methylco tumors: C- 20th weeks in group –
[49]. tional Group1 anthrene, Fibrosarcomata 0.0088mM C: 18cm2 - 27cm2 (12th
L-
1977 animal (control, C, 40mg, SC , liposarcomata week) - 22cm2.
0.00026
study. n=20) – for mM L: 10cm2 – complete
[TD$INLE] Animal Group2 (low tumor H- regression(one half of original
model – dose, L induction 0.88mM tumor size).
guinea n=20) in all H: 18cm2 – 50cm2.
pig Group3 high animals. Dose
dose H, reversal Tumour histology H: compactly arranged
n=20). after sarcoma cells surrounded by a
4week
interval:
delicate stroma.
C- C: haemorrhagic connective
0.88mM, tissue.
L- L: wide spaces lined by
0.88mM, PAS positive material,
H- containing RB
0.00026m
M

leukemic cell lines by 10 mM vitamin C (VC) when used solo or
in combination with 10 mM vitamin K3 (VK3) (100:1) for a
period of 24 h and attributed the causative mechanism to O2*/
H2O2 formation and mitochondrial membrane depolarization.
The formed H2O2 induced the activation of transcription factor
NF-kB,30 which is responsible for transcription of tumour
suppressor genes like p53 that in turn transcribed pro-
apoptotic genes such as PUMA, Noxa, Bim, Bad, Bax and
evoked apoptosis in the cell lines via caspase-3 protease
activation.31 Furthermore the Jurkat cell line was found to be
more resistant to VC or VC and VK3 (100:1) than K562 cells that
were highly susceptible to the pro-oxidant agents at much
lower concentrations. But a 1000:1 ratio has shown to induce
late apoptotic events and mitochondrial depolarization in
cells. In addition, the pro-oxidant mixture has shown to
activate c-jun transcription, possibly resulting from the
crosstalk between the signalling pathways.32 On the other
side normal lymphocytes strikingly did not show changes to
any concentration of the VC or pro-oxidant mixture that can
be explained by presence of efficient H2O2 scavenging
mechanisms.8,28
Another study done by Deubzer et al.33 in neuroblastoma
cell lines with a 48 h incubation period with ascorbate showed
that the preferential cytotoxicity of ascorbate in these cells
was facilitated by high ferritin content, typical of these cells,
which is an iron source for generation of ROS (O2*) by
pharmacologic ascorbate. The O2* generated was shown to
release iron from ferritin in turn that kept the process ongoing
and also generated H2O2 by reacting with H+. This H2O2 was
responsible for oxidative damage by inducing DNA strand
breaks in the cells and mediated cell death.34 In addition, high
rate of aerobic glycolysis occurring in the cell lines was found
to contribute to acidic microenvironment (lactate production)
that facilitates further iron release from ferritin. The results of
this study showed a reverse correlation between ascorbate
cytotoxicity and concentration of cells in the culture,
suggesting increased cellular capacities to detoxify H2O2 with
increasing cell concentrations. One more study included in
 archives of oral biology 58 (2013) 563–574
this review is that of Carosio et al.,35 which also evaluated the
pro-oxidant concentrations of sodium ascorbate with a
treatment period of 24 h in five neuroblastoma cell lines,
showed them to be highly susceptible to 3 mM doses of
ascorbate.
The study carried out by Chu et al.24 analysed the
cytotoxicity of sodium ascorbate (Sod.asc) (sodium salt of
ascorbic acid) propagated through DNA fragmentation and
caspase activation in human oral squamous cell carcinoma
and promyelocytic leukaemia cell lines after an incubation
period of 48 h with Sod.asc and attributed it to the pro-oxidant
property of pharmacologic VC (2–4 mM). The study tested
three herbal extracts: Drynaria baronii (DB), Angelica sinensis
(AS) and Cornus officinalis (CO) of which two (DB and CO) have
shown to synergistically enhance the cytotoxicity of 1 mM VC,
but the exact mechanism for this synergistic enhancement of
VC’s cytotoxicity was not explained.
Belin et al.36 studied the effects of pharmacologic AA on Raji
cells in vitro with an experimental treatment period of 96 h
and reported decrease in cell density or cell proliferation,
percentage of viable cell with increase in pro-oxidant dose and
exposure time of AA. The author proposed that AA inhibits cell
division and further promotes necrosis by down-regulating
the expression of genes necessary for S-phase progression in
actively proliferating cells that are susceptible to AA treatment
rather than the quiescent cells.
Kishino et al.25 tested the effects of pharmacologic levels of
sodium 5,6-benzylidene-L-ascorbate (SBA) in human oral
squamous cell carcinoma and human myelogenous leukae-
mia and normal oral cell lines incubated for 48 h in vitro and
observed a profound anti-tumour potential activity of SBA
against carcinoma cell lines. SBA, a derivative of AA, induced
cytotoxicity was presented in the form of autophagic cell
death during the early exposure periods followed by necrotic
and apoptotic changes in squamous cell carcinoma and
leukaemia cells respectively as analysed by evident DNA
fragmentation or intranucleosomal DNA fragmentation
and ultrastructural changes in the cells. Therefore, the
pro-oxidant activity is shown to be a dose-dependent
phenomenon, not only of AA but also its derivative com-
pounds like SBA.
A study done by Chen et al.37 evaluated the cytotoxic effects
of high doses of ascorbate on normal cells and tumour cell
lines like melanoma and lymphoma with exposure time of 2 h
followed by cell culturing for 24–48 h in absence of ascorbate.
The study reported that EC50 (concentration of ascorbate that
reduced survival by 50%) for normal cells was >20 mM
suggesting their strong resistance to high doses of ascorbate,
as high as 20 mM; while lymphoma cells were the most
sensitive to comparatively lower pharmacologic doses (1 mM)
followed by melanoma cells (8 mM). This variation in
sensitivity may be attributed to the complex networks on
which H2O2 acts, in combination with the spectrum of
functional mutations intrinsic to each cancer cell line that
are not present in normal cells.
A separate study done by Chen et al.15 measured the
ascorbate radical and H2O2 concentrations in blood and
extracellular fluid (plasma) at 30 min interval following its
high dose administration via three routes (IV, IP, Oral) in rats.
The ascorbate radical and H2O2 concentrations were found to
571
be high in ECF and comparatively very low in blood, suggesting
that these products would be immediately destroyed by
plasma and red blood cell proteins, thus satisfying the concept
of pharmacologic ascorbate in vivo serving as a prodrug for
selective delivery of H2O2 to the extracellular space. Further-
more, the achieved concentrations of these oxidative sources
were high with intravenous route, suggesting that parenteral
administration bypasses the tight control mechanisms of
ascorbate in humans.38–40 Therefore parenteral route of
administration of ascorbate would obtain millimolar concen-
trations in ECF and blood with preferential generation of Asc*
and H2O2 in ECF but not blood. A similar result was obtained in
another study by Chen et al.41 where the fraction of lymphoma
cells undergoing death was decreased in presence of added
human RBC in vitro, suggesting that blood would protect
tumour cells from ascorbate mediated cell death.
Bhat et al.42 did a study on human lymphocytes testing the
mechanism of pro-oxidant activity of AA ex vivo with
incubation period of 90 min, in which endogenous copper in
the lymphocytes was utilised for oxidative DNA breakage,
which is in support of the concept of free radical generation
induced cytotoxicity in presence of transitional metal ions (in
this case – copper ions), a putative mechanism for anticancer
properties of AA. This was proved by the administration of
neocuproine (NC), a Cu(I) sequestering agent, where increased
concentrations of NC decreased DNA breakage, suggesting the
necessary presence of copper ions for generating free radical
induced oxidative stress.
A study by Kinoshita et al.43 showed that higher concen-
trations (0.5–2 mM) of sodium ascorbate when treated for 24 h
enhanced growth inhibitory effects of dental metals like Cu
and Pd alloy on human squamous cell carcinoma, subman-
dibular carcinoma cell lines in vitro, while low doses of
ascorbate (0.25 mM) significantly reduced this effect. In
addition, these metals have shown to increase the radical
intensity of ascorbate in the early period of 30 min, suggesting
the modulation of biological behaviour of dental metals by
ascorbate in a dose dependent fashion.
Sakagami et al.44 also evaluated the pro-oxidant effects of
sodium-L-ascorbate in human oral tumour cell lines with
incubation period of 24 h in vitro in which H2O2 and radical
production depended on the pH of the medium showing
higher intensities at higher alkaline pH (9–10) compared to
that of physiological pH (7.2–7.4). The mechanism behind
radical intensity and pH was not explained.
Vojdani et al.45 examined effects of different doses of AA
in vivo human blood leukocytes in which doses as high as
5000 mg/day for about 2 weeks neither affected the viability of
cells, natural killer cell cytotoxic activity nor levels of DNA
adducts. This ensures that pharmacologic doses of AA can be
safely administered in vivo without any debilitating effects on
normal blood cells.
The study of Kelley et al.46 on human oral squamous cell
carcinoma cell lines in vitro showed that a pro-oxidant
combination of iron and ascorbate when administered for
15 min increased the efficacy of a photodynamic therapy of
cancer (Photofrin) by greatly increasing the production of
membrane-derived lipid free radicals, which led to in-
creased photosensitization of cancer cells. This further led
to lipid hydroperoxidase formation, which increased the
572 archives of oral biology 58 (2013) 563–574
cell’s susceptibility to iron-induced Fenton chemistry that
resulted in a net significant reduction in the surviving cell
fraction.
An in vitro study done by Noto et al.47 in human oral
epidermoid cell lines reported VC in combination with VK3,
after 1 h of incubation significantly inhibited the growth of
tumour cells well fitting in the concept of hydrogen peroxide
formation by both these vitamins.48
Harada et al.26 published an in vivo animal study on
hamsters, where diethylnitrosamine (DEN) was used to induce
oral leukplakia and tested the simultaneous supplementation
of 1% AA for an experimental period of about 58 weeks; in
which the supplemented group of hamsters had a significantly
lower percentage of incidence of leukoplakic lesions in
comparison to the control group, suggesting that AA supple-
mentation has potential antiproliferative effect due to its
potent pro-oxidant activity.
Migliozzi49 reported an in vivo animal study in guinea
pigs, in which tumours of fibrosarcomata and liposarcomata
were artificially induced and supplementation of various
doses of AA for about 20 weeks was evaluated for their
effects on the tumours. Supplementation of a high dose of
AA (1 g/kg/day or 0.88 mM) in a previously low dose
maintained group (0.3 mg/kg or 0.00026 mM) showed a
complete regression of tumours by 20th week of experi-
ment, suggesting a potential antitumour and antiprolifera-
tive activity of pharmacologic AA as a pro-oxidant, whereas
the tumour size increased in a previously high dose
maintained group (1 g/kg) when the dosage was reversed
to 0.3 mg/kg.
4.1. Outcomes
All the included studies of this review have evaluated the
pro-oxidant effects and pro-oxidant concentrations of
pharmacologic ascorbic acid that resulted in potent anti-
tumour and anti-proliferative actions on tumour cells. The
important outcomes of this systematic review are as
follows: The optimum concentration of ascorbic acid used
to produce potential pro-oxidant associated cytotoxicity
was found to be 3–5 mM in vitro, 0.88–5 mM in vivo animals,
0.5–2 mM ex vivo in humans. These pharmacologic con-
centrations were found to be cytotoxic to tumour cells,
mediated by various mechanisms like induction of apopto-
sis (caspase activation), necrosis, free radical formation,
H2O2 generation, DNA fragmentation. In contrast, the same
pro-oxidant concentrations had no effect the normal cells.
The average AA incubation time employed in most of the
in vitro studies was about 24–48 h while a human in vivo
study used an experimental period of 2 weeks45 and another
human ex vivo study used a treatment period of 90 min.42 A
couple of animal studies demonstrated a treatment period
58 weeks26 and 20 weeks.49 The EC50 of Ascorbic acid was 1
to <2 mM for most of the tumour cell lines, while EC50 in
normal cell lines was found to be >20 mM. The potential
pro-oxidant associated cytotoxic activity of AA towards
tumour cells along with its striking unaffecting behaviour
towards the normal cells provides us an implication for its
promotion in the therapy for oral neoplasms with substantial
benefits.
5. Conclusion
The results of our systematic review show that the
pro-oxidant activity of pharmacologic ascorbic acid is a
part of its dose-dependent bimodal activity and is a result of
the proposed Fenton mechanism. In vitro, animal and
ex vivo studies of pharmacologic AA have yielded meritori-
ous results proving vitamin C as an effective cytotoxic
agent against oral neoplastic cells with potentially no
harming effects on normal cells. However, a shortage of
clinical trials and in vivo human studies pertaining to
evaluation of anti-tumour activity of vitamin C in tumours
of oral cavity remains a lacuna in concluding ascorbic acid
as a beneficial therapeutic option in treatment of oral
neoplasms.
5.1. Implications for practice and research
A next step assessment and evaluation of the pro-oxidant
effects and pro-oxidant concentrations of vitamin C in
tumours of oral cavity, in vivo humans would fill in the space
of the research done so far and would bring in its ultimate
practical application of Oral health. Like the two-faced Janus,
vitamin C exhibiting its selective cytotoxicity towards tumour
cells, could serve a worthy, natural therapeutic option in
treating the tumours of oral cavity.
5.2. Limitations of the review
We acknowledge the presence of publication bias in this
review. The employed parameters for evaluation in all the
studies are not homogenous; therefore the review proceeded
as a heterogenous study. If methods and modes of evaluating
the pro-oxidant activity could be more standardized with
minimal data set it would help by providing homogenous data
for systematic reviews in the future.
Conflict of interest
No conflict of interest.
Funding
No external sources of funding.
Ethical approval
The manuscript submitted is that of a review type. Scientific
Review Board approval accomplished.
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