Journal of Cancer Therapy, 2013, 4, 1366-1372 Published Online October 2013 (
Megadoses of Sodium Ascorbate Efficiently Kill HL60
Cells in Vitro: Comparison with Arsenic Trioxide
Domenico Mastrangelo1, Lauretta Massai1, Giuseppe Fioritoni2, Antonio Iacone2,
Paolo Di Bartolomeo3, Patrizia Accorsi3, Tiziana Bonfini3, Michela Muscettola1, Giovanni Grasso1
1Department of Medical, Surgical, and Neurological Sciences, University of Siena, Siena, Italy; 2Pescara Cell Factory Foundation,
Pescara Main Hospital, Pescara, Italy; 3Department of Hematology, Pescara Main Hospital, Pescara, Italy.
Received September 23rd, 2013; revised October 17th, 2013; accepted October 23rd, 2013
Copyright © 2013 Domenico Mastrangelo et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Arsenic Trioxide (ATO) is widely acknowledged as the treatment of choice for Acute Promyelocytic Leukemia (APL).
It is a “two-sided” drug since it can induce differentiation or kill APL and other tumor cells according to the dosage.
Part of the cytotoxic effects of ATO on APL cells is due to its pro-oxidant activity, a characteristic which ATO shares
with a number of other compounds, including high doses of ascorbate (ASC). In a comparative investigation on the cy-
totoxic effects of both ATO and ASC on HL60 (APL) cell lines, in vitro, we have been able to confirm the known cy-
totoxic effects of ATO, but, more importantly, we have demonstrated that ASC is significantly more effective than
ATO, in killing these cancer cells in vitro, when the concentrations are maintained within the millimolar (mM) range,
i.e. the range of plasma concentrations at which ASC induces oxidative damage to tumor cells. Since these plasma lev-
els can be reached only by the intravenous administration of high doses of ASC, we propose that intravenous high doses
of ASC may represent a potentially revolutionary new approach in the management of APL.
Keywords: HL60; Acute Promyelocytic Leukemia; High Doses of Ascorbate; Cell Count and Viability Assays
1. Introduction
Acute Promyelocytic Leukemia (APL) is a rare, though
extremely malignant subtype of acute myeloid leukemia
(AML), which, only a few decades ago, Hillestad de-
scribed as characterized by “a very rapid fatal course of
only a few week’s duration” with a white blood cell
(WBC) picture dominated by promyelocytes and a severe
bleeding tendency [1,2].
The use of all-trans retinoic acid (ATRA) as a differ-
entiating agent is well established, in the treatment of
APL [3], but in the last two decades or more, Arsenic
Trioxide (ATO), initially proposed by Chinese authors as
the first line treatment of APL [4], has been largely ac-
knowledged as the “most effective single agent” [5], “the
most biologically active single drug” [6], and more re-
cently, “a drug from a poison” to be used not only in the
treatment of this leukemia, but also in other types of
cancer [7], for its efficacy, cost effectiveness, and low
toxicity [8,9].
Studies on HL60 (human promyelocytic leukemia) cell
line, have shown that ATO can be viewed as a two-
sided drug, inducing either differentiation or apoptosis on
these cell lines [10], according to the dosage employed.
These data have been confirmed by studies on retino-
blastoma cell lines, showing that in low doses (1 μM) it
induces differentiation, while in high doses (2 μM) it
behaves as a pro-apoptotic drug and effectively inhibit
tumor formation in vitro [11].
As reported by different Authors [12-14], oxidative
stress is critical to the ATO-induced apoptotic process,
with generation of ROS, including, among others, H2O2,
which is considered a metabolite of the drug [15]. Inter-
estingly, ATO shares this pro-oxidant activity with a
number of other compounds, including sodium ascorbate
(ASC) administered in high doses by intravenous injec-
tion [16-20].
Given the above mentioned similarities underlying the
anticancer activity of ATO and ASC, and the high cyto-
toxic potential shown by ASC in different cancer cell
lines in vitro [16-21], we have undertaken the present
investigation in order to assess the cytotoxic effect of
ASC on HL60 cells in vitro in comparison with ATO,
which is the drug of choice in the treatment of APL.
Copyright © 2013 SciRes. JCT
Megadoses of Sodium Ascorbate Efficiently Kill HL60 Cells in Vitro: Comparison with Arsenic Trioxide 1367
2. Materials and Methods
HL60 (Human Promyelocytic Leukemia) cell lines were
kindly supplied by the Department of Molecular and
Developmental Medicine of the University of Siena. All
reagents, including culture media, Sodium Ascorbate
(ASC), Trypan Blue, Hoechst 33342, and Propidium
Iodide (PI), were purchased from Sigma-Aldrich. Arse-
nic trioxide (ATO chemical formula: As2O3) vials (Tri-
senox) were kindly supplied by the Department of Hae-
matology of the Pescara main hospital (Pescara, Italy).
Automated cell count and viability was performed by
using the “Muse”™ (Merck-Millipore) automated cell
analyzer. A Zeiss Axioplan2 microscope was used for
fluorescence microscopy and a Shandon cytocentrifuge
for microscopic/morphologic analysis of cell suspen-
Cell counting, before and after exposure to increasing
doses of either ATO and ASC was performed with both
the manual (Trypan Blue Exclusion Test) and automated
(“Muse”™) methods. The automated method, using the
“Muse”™, was carried out according to the instructions
supplied by the manufacturer which encompass an in
house method of nuclear staining for the assessment of
cell viability. The Trypan Blue Exclusion Test was per-
formed according to the standard procedures [22,23]. At
the end of each count and viability test HL60 cell suspen-
sions were stained with Hoechst/PI as described else-
where [24] and aliquots of about 1 × 105 cells were de-
posited onto alcohol-washed glass microscope slides by
using a cytocentrifuge, at 1000 r.p.m. for 5 minutes, and
then observed under fluorescence microscopy.
HL60 human promyelocytic leukemia cells were
grown in RPMI supplemented with antibiotics, glutamine,
and 20% FBS, at 37˚C in 5% CO2/95% air. During the
phase of exponential growth, the cells were harvested
and counted with the “Muse”™ automated cell counter/
analyzer and then diluted to a concentration of about (2 -
5) × 105/ml.
Four dilutions of 2, 4, 6, and 8 μg/ml of ATO were
used, starting from a stock solution of ATO commercial-
ly available at a concentration of 1mg/ml (Trisnox™), by
simply adding 2, 4, 6, and 8 μl, respectively, of Tris-
enox™, to 1 ml of culture medium containing (2 - 5) ×
105 HL60 cells. These concentrations were chosen ac-
cording to the data reported by Yediou and coll. [10].
A 1 M solution of ASC was prepared fresh each time
as described elsewhere [21], and aliquots of 1, 3, 5, and 7
μl of this stock solution were added to four different
wells containing 1ml each of culture medium in which (2
- 5) × 105 HL60 cells were suspended, to a final concen-
tration of 1, 3, 5, and 7 mM, respectively. ATO can be
stored at room temperature for up to 36 months.
Aliquots of 1 ml of culture medium containing (2 - 5)
× 105 HL60 cells were loaded into each of 12 well plate,
and added with the four different concentrations of ATO
(2, 4, 6, and 8 μg/ml); the same procedure was used for
ASC (1, 3, 5, and 7 mM). The cells were exposed for a
total of 18 - 24 hours. One control (no treatment) sample
was also included. At the end of the incubation period the
cells were collected in vials, and mixed with the Muse™
Count & Viability Reagent, according to the procedure
supplied by the manufacturer, for automated counting
and viability analysis. Namely, 10 μl of each sample
were added to 190 μl of the Muse™ Count & Viability
Reagent, which differentially stains viable and non-vi-
able cells based on their permeability to the two DNA
binding dyes present in the reagent. A specific Software
Module then performs calculations automatically and
displays data in two dot plots as shown in Figure 1.
Each experiment was repeated at least twice, and a to-
tal of six experiments was carried out, with the auto-
mated (“Muse”™) cell counting and viability tests, with
and without (control sample) increasing doses of ATO
and ASC, as reported. The results have been cumula-
tively analyzed by calculating the mean, standard error,
standard deviation, and 95% confidence intervals (95%
CI) of the percentages of living cells for each experiment.
The statistics was performed by calculating the two-
tailed “p” value of the difference between the mean per-
centage of viable cells in each of the four doses of ASC
(1, 3, 5, and 7 mM) as compared to ATO (2, 4, 6, and 8
Aliquots of cells were also stained with Hoechst/PI,
according to the standard protocols, deposited onto glass
microscope slides with a cytocentrifuge, and finally ob-
served under fluorescence microscopy [25]. For statisti-
cal analysis, the mean values and related 95% confidence
intervals were calculated for each set of repeated meas-
urement by using the SPSS statistical package, version
3. Results
The results of this experiment are reported in Table 1
and summarized in the diagram of Figure 2. As shown in
the graph, the mean percentages of live HL60 cells (the
“y” axis) proportionally decrease by increasing the con-
centrations of both ASC and ATO (the “x” axis). How-
ever, at any given concentration, ASC always kills more
HL60 cells than ATO. The two tailed “p” value of the
difference in the percentage of live cells between ASC
and ATO suggests that ASC 1, and 3 mM is more effec-
tive than ATO 2, and 4 μg/ml, respectively, in reducing
the survival of HL60 cells in culture even though the
difference does not appear statistically significant. How-
ever, at 5 mM, ASC is significantly more effective (p =
0.0038) than ATO (6 μg/ml) and the difference becomes
highly statistically significant (p = 0.0001) in favor of
Copyright © 2013 SciRes. JCT
Megadoses of Sodium Ascorbate Efficiently Kill HL60 Cells in Vitro: Comparison with Arsenic Trioxide
Copyright © 2013 SciRes. JCT
Figure 1. A representative sample of the cell viability profile obtained by treating HL60 cells with ASC (left column of dia-
grams) and ATO (right column) according to the “MUSE”™ cell analyzer. The upper left set of diagrams is referred to the
control (untreate d) sample. As shown in the diagram, both ASC and ATO effic iently kill HL60 cells in vitro, but, at any given
concentration, ASC always kills more c ells than ATO, and this difference is statistically significant in favor of ASC, particu-
larly at 5 and 7 mM (dose 3 and 4).
Megadoses of Sodium Ascorbate Efficiently Kill HL60 Cells in Vitro: Comparison with Arsenic Trioxide 1369
Figure 2. Comparison of the mean percentages of viable
HL60 cells (“y” axis) after treatment with ASC (blue bars)
and ATO (red bars). The green bar indicates the untreated
(control) cells. The statistical analysis of these data reveals
that at “dose 3” (ASC 5 mM and ATO 6 µg/ml) and “dose
4” (ASC 7 mM and ATO 8 µg/ml) ASC is significantly more
efficient than ATO in killing HL60 cells in vitro.
Table 1. Mean percentages of viable cells in the untreated
(control) and treated (ASC and ATO) HL60 cells, at dif-
ferent dosages, in six consecutive experiments.
EXP.N. Control
(% vital) DOSE ASC
(% vital)
(% vital)
1 96.5 1 91.9 93.2
2 61.5 74.8
3 19.9 59.7
4 15.4 44.1
2 98 1 92.4 92.2
2 34 70.7
3 10.6 58.9
4 8.5 32.7
3 97.4 1 89.3 73.2
2 48.3 28.5
3 30.6 21.7
4 2.2 21.9
4 97.2 1 82 93.1
2 13 81.8
3 4.3 62.5
4 2.2 45.4
5 97.2 1 84.3 95.2
2 59.3 86.5
3 34.4 69.8
4 8.6 39.2
6 91.2 1 70.3 94.3
2 15.5 86.8
3 11.7 65.8
4 5.8 50.1
ASC when ASC 7 mM is compared to ATO 8 μg/ml. The
LC50 for ATO is above 6 µg/ml, as reported by Yediou
and coll.10, while for ASC it is in between 3 and 5 mM,
as reported by different Authors [16-21].
The Trypan Blue analysis, even if not informative re-
garding the apoptotic condition of treated HL60 cells,
confirmed the data obtained with “Muse”™.
Regarding the Hoechst/PI staining, as shown in Figure
3 the cellular changes induced by ATO seems to be sub-
stantially different from those obtained with the exposure
to millimolar (mM) doses of ASC. Namely, while ATO,
at the maximum dosage tested herein, clearly shows
signs of apoptotic cell damage, encompassing, nuclear
fragmentation and chromatin condensation, ASC treated
HL60 cells are clearly necrotic, as shown by the red color
(a) (b)
(c) (d)
Figure 3. Hoechst/PI fluorescent microscopy staining of
untreated (controls) and treated (ASC and ATO) HL60
cells. 400× magnification. Untreated (control) cells (a) ap-
pear uniformly blue with nuclei show ing a loose chr omatin,
and no signs of necrosis or pyknosis. This feature is com-
patible with a well preserved viability, as also confirmed by
the “MUSE”™. The same cells, treated with 3 and 5 mM
ASC ((b) and (c) respectively), appear uniformly stained in
red (PI) thus indicating an advanced necrotic condition. On
the contrary, HL60 cells treated with ATO at 4 and 6 µg/ml
((d) and (e) respectively), are almost uniformly stained in
blue, but the condensed chromatin and nuclear fragmen-
tation are indicative of early/late stage apoptotic condition.
Copyright © 2013 SciRes. JCT
Megadoses of Sodium Ascorbate Efficiently Kill HL60 Cells in Vitro: Comparison with Arsenic Trioxide
of the nuclei which were stained by PI, but not by Heo-
chst 33342. These results confirm our previous observa-
tion of the effects of mM doses of ASC on Y79 retino-
blastoma cells in vitro [21].
4. Discussion
Historically, four periods have been identified, in the
treatment of APL, which have marked a progressive im-
provement in the cure of this disease, namely: 1) chemo-
therapy (CT) alone (with antracyclines and Ara-C), from
1957 to 1985; 2) all-trans retinoic acid (ATRA), intro-
duced in 1985; 3) Arsenic Trioxide (As2O3-ATO) intro-
duced in the mid 1990s; 4) Combination of ATRA and
ATO, in 1998. With all these achievements, APL has
passed from a 5-year disease-free survival (5yDFS) of
about 35% - 45%, to a 6 year disease-free survival
(6yDFS) of 86% (±10%) [26].
The role of ROS, H2O2, redox potential, and oxidative
stress in the pro-apoptotic, anticancer activity of ATO,
further illustrated in detail by de Thé & coll. [27], Selva-
raj & coll. [28], Sanchez & coll. [29], among others, sug-
gests that drugs with oxidizing potential may represent
the new frontier in cancer treatment [30], especially if we
consider that cancer cells have more commonly exhaust-
ed their antioxidant machinery [31] and therefore are
more vulnerable to oxidizing compounds.
ASC (Vitamin C) was first isolated in 1928 by the
Hungarian biochemist Dr. Szent-Gyorgyi who received
the Nobel Prize for this discovery in 1937 [32].
The history of ASC as an anticancer molecule is very
controversial [33]. McCormik, nearly 60 years ago, sug-
gested that ASC protects against cancer by increasing
collagen synthesis [34,35] while Ewan Cameron hypo-
thesized that ASC could have anti-cancer effects by in-
hibiting hyaluronidase and thereby preventing cancer
spread [36]. Although successful clinical trials had been
reported, by Cameron and the twofold Nobel laureate
Linus Pauling, on terminal cancer patients, Charles Mo-
ertel, at Mayo Clinic, reported negative results, and his
trials were credited as the definitive proof of the ineffi-
cacy of ASC in treating cancer [36,37]. In Moertel’s trial,
however, ASC was given orally, while Cameron and
Pauling had used both the intravenous and oral route of
administration simultaneously. Report survey data on
intravenous ASC show that ASC is used in doses up to
200 g per infusion, for a variety of pathological condi-
tions, with very few adverse effects [38], and that, when
taken orally, plasma ASC concentrations never raise be-
yond the level of 100 μmol/l, due to the limited bioavail-
ability of the molecule and renal excretion [39]. On the
contrary, when administered by intravenous infusion,
ASC reaches plasmatic concentrations in the order of
millimolar range, which would never be obtained by the
administration of oral doses [18-20,40,41], and behaves
as a very powerful oxidant, suitable for the therapeutic
use in cancer [42].
The anticancer effects of ASC have been widely dem-
onstrated in a number of different tumor cell lines [17-
20], and phase I clinical trials have been already per-
formed, in different cancers, showing that, even in very
high doses, it is well tolerated [43] and has the potential
to selectively kill cancer cells due to its capacity of pro-
ducing intracellular H2O2, with oxidation of different
cellular components, and cell death [17-20,31,40].
Although the use of ASC in the treatment of APL has
been already proposed by both Yedjiou & coll. [10] and
Bachleitner-Hofmann and coll. [44], in combination with
ATO, ASC has never entered in the routine treatment of
APL; this can be explained by the substantial difference
in the dosage proposed by both Yedjiou and Bachleitner,
when compared to our protocol. In fact, while it has been
observed that adding ASC in the treatment of APL may
slightly increase the cytotoxic potential of ATO, the dose
of ASC recommended by both Authors, never over-
comes 150 µM, which is within the range of plasma
concentrations that can be reached by oral administration
of ASC. More importantly, at plasma concentrations in
the range of micromoles (µM), ASC behaves as an “anti-
oxidant”, and, as such, it has also been reported to protect
HL60 cells from arsenic toxicity [45] and antagonize the
toxic effects of chemotherapeutic agents [46]. On the
contrary, at concentrations in the order of magnitude of
the millimoles (mM), used in the present investigation, it
clearly shows pro-oxidant effects, on tumor cells, which
lead to an increased production of H2O2 and other ROS
and consequent oxidative damage of different cell struc-
tures, as reported by several Authors [17-20,31,40].
The data reported herein substantially confirm the ef-
fectiveness of ATO in the treatment of APL, but add new
relevant information, by showing that ASC, when used in
mM concentration, as those that can be reached by intra-
venous injection of pharmacologic doses, is significantly
more effective than ATO in killing HL60 cells in vitro.
The advantages of ASC, as compared to ATO, in the
treatment of APL are clear:
1) In phase I clinical trials on metastatic pancreatic
cancer [43], ASC has been administered at doses varying
from 50 to 100 grams per infusion, three times a week
for eight weeks, with plasma levels up to 30 mM and no
major side effects and;
2) Due to its peculiar “metabolic” effect, ASC has
been widely reported to selectively kill cancer cells, be-
ing substantially harmless for normal cells.
Given all the above, the Authors strongly believe that
high (“pharmacologic”) doses of ASC may represent a
second revolution in the treatment of APL, and suggest
to start a phase I-IV clinical investigation on ASC in the
treatment of selected cases of APL, in order to verify its
effectiveness in the treatment of this leukemia.
Copyright © 2013 SciRes. JCT
Megadoses of Sodium Ascorbate Efficiently Kill HL60 Cells in Vitro: Comparison with Arsenic Trioxide 1371
5. Acknowledgements
The authors thank Dr. Felice Arcuri, of the Department
of Molecular and Developmental Medicine of the Uni-
versity of Siena (Italy), for contributing to this work by
supplying HL60 cell lines.
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