Effects of Sodium Ascorbate (Vitamin C) and 2-Methyl- 1,4-

Naphthoquinone (Vitamin K3) Treatment on Human

Tumor Cell Growth In Vitro
1. Synergism of Combined Vitamin C and K3 Action

VINCENZO NOTO, MD,* HENRYK S. TAPER, MD,t JIANG YI-HUA, MD,*’$ JAAK JANSSENS, MD,§
JAN BONTE, MD,§ AND WILLIAM DE LOECKER, MD’

The effects of sodium ascorbate (vitamin C) and 2-methyl-1,4-naphthoquinone(vitamin K3) administered

separately or in combination on the in viiro cultured human neoplastic cell lines MCF-7 (breast carcinoma),
KB (oral epidermioid carcinoma), and AN3-CA (endometrial adenocarcinoma) have been examined. When
given separately, vitamin C or K3 had a growth inhibiting action only at high concentrations(5.103 /.tmol/
1 and lo5 nmol/l, respectively). Combined administration of both vitamins demonstrated a synergistic
inhibition of cell growth at 10 to 50 times lower concentrations. At this level separately given vitamins
are not toxic. The sensitivity to this treatment was somewhat different in the three cell lines, being slightly
higher for KB line. This tumor cell growth inhibitory effect was completely suppressed by the addition
of catalase to the culture medium containing vitamins C and K3, suggesting an excessive production of
hydrogen peroxide as being implied in mechanisms responsible for the above-mentionedeffects.
Cancer 63:901-906, 1989.

A MONG CHEMICAL co:mpounds attracting great in-

terest in cancer prevention and treatment, several
vitamins have been examined. Thus, ascorbic acid (Vit
C) has been shown to inhibit the formation of carcinogenic
and mutagenic compounds in foods and has been sug-
gested to be: useful in the ]prevention of gastric cancer.’
At nontoxic concentrations,,Vit C potentiates the growth
inhibitory effect of certain agents such as 5-fluorouracil,
bleomycin sulfate, sodium butyrate, cyclic AMP-stimu-
lating agents, and x-irradiation on neuroblastoma cells.*
Ascorbate also is known to potentiate the cytotoxicity of
misonidazole3 and of 6-hydroxydopamine on mouse
neuroblastoma cells: on human neuroblastoma cells in
vitro,’ and of 3-arnino-l,2,,4,-triazole(ATA) on Ehrlich
ascites carcinoma cells.6 Vitamin C may accumulate in
From the *Afdelingen Biochemie, §Gynaecologische Gezwelziekten,
Katholieke Universiteit Leuven, Leuven, Belgium, ?Unit6 de Biochimie
Toxicologique et Canctrologique, UniversitC Catholique de Louvain,
Bruxelles, Belgium.
Supported by grants from the Belgian National Foundation for Medical
Research (FGWO), and the “Associazione Italiana per la Ricerca sul
Cancro (Milano)” (V.N.).
$ Current address: Department of Obstetrics and Gynecology, First
Hospital, Beijing Medical University, China.
Address for reprints: William De Loecker, MD, Afdeling Biochemie,
Campus Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium.
Accepted for publication September 15, 1988.
the tumors thus decreasing its level in peripheral blood
of some cancer bearing subje~ts.’,~ In vitro Vit C may
even reverse malignant cell transformation as demon-
strated on C3H/ 1OTYz mouse embryo cells transformed
by 3-methylcholanthrene,’ or may demonstrate a cyto-
toxic action towards different tumor cell^.^^^^^^^' I
Similarly it has been described that vitamin K3inhibits
the growth of different types of mammalian tumor cells
in culture.’* It increases the thermosensitivity of Ehrlich
ascites carcinoma cells and enhances in vitro the antineo-
plastic activity of 5-fluorouracil in Friend murine eryth-
roleukemia cells, and of methotrexate in tumor-bearing
anirnal~.’~ ~ administration of vitamins C and K3
In’ ’vivo
combined to hepatoma bearing mice, results in a poten-
tiating effect of chemotherapy at cytotoxic dose levels
proportionally lower than used in human cancer treat-
ment. l 5
In the present investigation the combined effects of vi-
tamins C and K3 have been analysed in vitro on human
cancer cell lines.
Materials and Methods
Sodium ascorbate (Vit C) and 2-methyl- 1,Cnaphtho-
quinone (menadion sodium bisulfite, Vit K3) (Sigma, St.
Louis) were dissolved in Eagle’s minimum essential me-

90 1
902 CANCERMa?*chI 1989
I T
-
Y
- 150
v)
m T
L
W
n
+
c
W
+
6 100
"
2 4 6 0 12 16 20
DAYS
FIG.1. Growth curve of MCF-7, KB and AN3-CA cell lines. Approx-
imately 1 X lo6MCF-7 (0),KB (A) and AN3-CA(0)cells were aliquoted
into separate T-25 plastic flasks (Nuclon, Nunc) and maintained in MEM
medium with 5% Nutridoma (Boehringer). After different time intervals
the cells were harvested and DNA content determined. Each value rep-
*
resents the mean of four experiments SEM.
dium (MEM) (Gibco, Grand Island, NY) and sterilized
by filtration through a 0.20-pm syringe filter (Gelman
Sciences, Inc., Ann Arbor, MI).
Catalase from Aspergillus niger (EC 1.1 1.1.6) (Sigma)
suspended in 3.2 mol/l (NH4)$304was dialyzed exten-
sively in Dulbecco's phosphate-buffered saline (PBS)
(Gibco) at 4°C. The concentration of the original stock
solution of catalase was 17 mg of protein/ml (Biuret) (7350
Sigma U/mg protein, one unit decomposing 1 pmol of
hydrogen peroxide per minute at pH 7.0 at 25°C).
The human cancer cell lines AN3-CA (human endo-
metrial adenocarcinoma) and KB (human oral epider-
moid carcinoma) (American Type Culture Collection,
Rockville, MD) and MCF-7 (human-derived breast car-
cinoma cell line) (Michigan Cancer Foundation, Detroit,
MI) were plated at a density of about 2. lo6per T-75 flask
(Nunclon,Nunc, Denmark) and maintained in air at 37°C
in a monolayer culture in growth medium consisting of
Eagle's MEM supplemented with 5% newborn calf serum
(Gibco), 2 mmol/l L-glutamine (Gibco), 1TOnonessential
amino acids (Gibco), 100 IU/ml penicillin and 100 pg/
ml streptomycin (Gibco),and 10 ng/ml crystalline insuline
(Sigma). The newborn calf serum was effectively stripped
to remove steroid hormones by a 30 minutes incubation
at 45°C in the presence of an equal volume of DCC sus-
pension (10 mmol/l Tris-hydrochloride (HCl), 1 mmol/l
ethylenediaminetetraacetic acid (EDTA), 0.5% activated
charcoal, 0.05% dextran (Grade C) (Sigma). In later ex-
periments, newborn calf serum was replaced by Nutri-
doma-SP (Boehringer, Mannheim, Germany) character-
ized by well-defined composition.
Vol. 63
When cells reached the stationary phase, the monolayer
was washed twice in Hank's basic salt solution (Gibco),
without magnesium and calcium, for 3 minutes, and
trypsinized for 30 secondes at 37°C with trypsin-EDTA
dissolved in phosphate buffered saline, pH 7.4 (1:250)
(Gibco). Cell clumps were disaggregated to create a sus-
pension of isolated cells by passage through a no. 25-gauge
needle. Cells were resuspended in fresh culture medium
and an appropriate number was used for subculturing or
for the experiments.
After trypsinization and dispersion, 1 X lo6 cells were
aliquoted into separate T-25 plastic flasks (T-25 Nunclon,
Nunc, Denmark) and maintained in MEM medium with
or without 5% newborn calf serum or with Nutridoma.
At about 50% of confluence, the different combinations
of vitamins were added for an incubation period of 1 hour,
then removed and replaced by fresh medium not con-
taining any added vitamins anymore. The vitamin con-
centrations amounted to 1 to lo4 pmol/l (0.198 pg/ml
to 1.98 mg/ml) for Vit C, and 10 to lo5 nmol/l(2.76 ng/
ml to 27.6 pg/ml) for Vit K3.When used in combination,
Vit K3 concentrations were always adjusted to 1/100 of
the Vit C concentration. Culture medium without vita-
mins was used as control. All tumor cells were harvested
when confluence was reached in the control untreated
series. Storage of the harvested cells took place at -20°C
until DNA was assayed. DNA determination was camed
out 1 or 2 weeks after the vitamin treatment, according
to the cell lines examined depending on the confluence
in the controls. The DNA determinations were performed
by colorimetric estimations using diphenylamine reac-
tion.16
The average DNA concentration expressed in micro-
grams per flask of the untreated controls was considered
as the base value and that obtained after Vit C and/or K3
administration was used for cytotoxic effect calculation.
The effects of Vit C and/or Vit K3 on the three human-
derived cancer cell lines were evaluated as growth inhi-
bition and calculated:
pg DNA/flask of control cells-pg DNA/
flask of treated cells
x 100
pg DNA/flask of control cells
Results
The addition to the medium of stripped newborn calf
serum or its replacement by Nutridoma did not effect the
outcome of the vitamin effects. The stationary phase as
well as the 50% confluence state were reached after dif-
ferent time intervals depending on the cell lines examined.
For AN3-CA and KB cells confluence was usually reached
after 4 to 8 days whereas for the MCF-7 cells up to 2
weeks had to be allowed (Fig. 1).
No. 5 SYIVERGISM O F COMBINED VIT c AND K3 ACTION * Noto et a/. 903

r a t i o n o f VIT C (p M o l a r )
10 lo2 lo3 2.10, 5.10, 10‘ 5.10‘ lo5 C o n c e n t r a t i o n o f VIT K, ( n M o l a r )
FIG.2. Inhibition of MCF-7 cell growth after the in vitro exposure for one hour to different concentrations of: vitamin C (0);vitamin K, (0)or
combined vitamins C and K3 (A).The cell growth inhibition was expressed as percentage of altered DNA content in treated cell culture related to
*
that of the untreated control culture considered as 100%.Each value represents the average SEM of four experiments carried out on different flasks
of cultured cells. Other details described in “Materials and Methods.”

Efpcts of Vitamins C or K3 Separately Administered Efects of Combined Vitamins C and K3

Vitamins C at lo3 pmol/l concentration (198.02 pg/
ml) produced on the MCF-7 cell line about 50% inhibition
(Fig. 2), whereas KB line required for a similar effect 5. lo3
pmol/l concentration (990.1 pg/ml) (Fig. 3) and AN3-CA
cells distinctly more than 5.10’ pmol/l (99.01 pg/ml)
(Fig. 4).
Vitamin K3 at concentrations exceeding 5. lo4 nmol/l
( 1 3.8 pg/ml) produced on IMCF-7 cells a 50% inhibition
(Fig. 2), whereas KB and ALN3-CAcell lines required for
a similar effect, levels in excess of respectively 5. 104nmol/
1 (Fig. 3) and lo4 nmol/l (2.76 pglml) concentrations
(Fig. 4).
In MCF-7 cells a 74% inhibition was obtained at Vit
C concentration of 500 pmol/l or 99.0 1 pg/ml and at Vit
K3 concentration of 5.103nmol/l or 1.38 pg/ml (Fig. 2).
When, however, dose levels of Vit C and Vit K3 were
added separately to growing MCF-7 cells the inhibitory
effects amounted to 28% and 2%, respectively. Higher
concentrations of combined Vit C and K3 increased pro-
gressively the percentage of growth inhibition of MCF-7
cell line up to 93%at the concentration of lo4 pmol/l and
1O5 nmol/l for Vit C and K3, respectively.
When separately tested at individual concentrations for
Vit C being 500 Fmol/l(99 pg/ml) and for Vit K3 5. lo3

-T
VI
100-
u
m -
Y
L 80-
0

c
-
FIG. 3. Inhibition of KB cell 0

growth by separate exposure to dif- E 60-

n
ferent concentrations of vitamin C
._
c
t
-
or K,, or combined both vitamins .-
LO-
C and Ks. Techniques and symbols f -
as in Figure 2 0

b 20-
s
- -
1 10 10’ 2.10‘ 5.10’ lo3 5.10, 10‘ Concentration o f VIT C ( p M o l a r )
10 lo2 lo3 2.103 5.10, 10‘ 5.10‘ 10’ Concentration o f VIT K, (nMolar)
904 CANCERMarch I 1989 Vol. 63

FIG.4. Inhibition of AN3-CA cell

growth by separate exposure to dif-
ferent concentrations of vitamin C
or KJ, or combined both vitamins
C and K3. Techniques and symbols
as in Figure 2.

1 10 lo2 2 lo2 5.102 lo3 5.103 10' Concentration o f VIT C ( p M o l a r )

10 lo2 lo3 2.10, 5.103 10' 5.10' lo5 Concentration o f VIT K, (nMolar)

nmol/l (1.38 pg/ml) practically no inhibition was observed Efect of Catalase

on the KB cell line. When, however, both vitamins were
At a concentration of 50 pg/ml catalase added to grow-
used in combination at the same concentrationsan almost
ing AN3-CA cells did not affect the growth properties of
50% inhibition was observed (Fig. 3). At Vit C levels of
this cell line. However, when AN3-CAcells were cultivated
lo3pmolll and Vit K3levels of lo4nmol/l used separately,
as described and simultaneously incubated for 1 hour with
no in vitro cytotoxic effects were seen on KB cell line,
catalase (50 pg/ml; 367.5 U/ml) and increasing levels of
whereas when these two vitamins were applied in com-
combined Vit C and K3, the previously observed growth
bination at the same concentrations, the percentage of
inhibitory effects of Vit C and K3assessed upon confluence
inhibition reached 100%.
of the control nontreated cell line had completely disap-
On the AN3-CAcell line Vit C and Vit K3administered
peared even at the highest vitamin concentrations ex-
separately, at concentrations of 200 pmol/l (39.6 pg/ml)
amined (Fig. 5).
and 2. lo3 nmol/l (0.55 pg/ml), respectively, were practi-
cally ineffective, whereas used in combination at the same
Discussion
concentrations an inhibition of 36% was observed (Fig.
4). A cytotoxicity of about 80% was reached when Vit C DNA determinations closely reflect the number of cells
and Vit K3 were used in combination at concentrations undergoing growth inhibition either due to an inhibition
of 500 pmol/l and 5. lo3 nmol/l, respectively. of cell division or/and cell death." It is shown that Vit C

FIG.5. Counteraction of catalase

(added at the dose of 50 pg/ml)
/ against the AN3-CA cell growth in-

2oz
+ hibitory effect of combined vitamins
.- C and K3 (0).AN3-CA cell growth
.- LO[ inhibition pattern was obtained by
combined vitamins C and K3 treat-
ment (0).Techniques as in preceed-
ing figures.
sm
lo2 2.102 5.102 lo3 5.10' 10' Concentration o f VIT C ( p M o l a r )
103 2.10' 5.10' 10' 5.10' lo5 Concentration o f VIT K, (nMolar)
No. 5 SYNERGISM OF COMBINED VIT
at relatively high concentra,tions produces a growth in-
hibiting effect on three human derived cancer cell lines.
This confirms previous studies on other mammalian cells
in culture and reveals that the concentration levels re-
quired for it cytotoxic effect differ from one cell line to
an~ther.~.~ . ~ ~ . ' ~K3when given alone, at below
Vitamin
noxious concentrations for the organism, appears virtually
harmless to the cancer cells. Combined administration of
Vit C and K3,however, produces synergistic cytotoxic
effects at low-dose levels. These potentiating compounds
belonging to the vitamins (only become noxious to the
organism at relatively high concentrations. Synergism here
is defined as a combined cytotoxic effect which exceeds
the sum of the individual cytotoxic effects.
Several hypothetical mechanisms may be involved in
the action of Vit C. The cytotoxicity induced by Vit C
seems to be primarily medliated by hydrogen peroxide
generated by its metabolicsilly oxidized form (dehydro-
ascorbate).6 It has also been suggested that the selective
toxicity of \'it C in tumor cells may be due to reduced
catalase levels in these cell:;, leading to cellular damage
through the accumulation of hydrogen p e r ~ x i d e . ~ . ~ . ~ specifically
The most established function of Vit K3 is its role in
regulating the synthesis of blood coagulation factors.'*,I9
However, Vit K3may also inhibit the binding of the epi-
dermal growth factor (EGF) to the membrane receptors,
cause membrane-lipid rearrangement and alter calcium
fluxes. 12,20The cytotoxic effects of many quinones, playing
an important role in oxidative phosphorylation, appear
to be mediated by a one-electron reduction to semiqui-
none radicals, subsequently entering redox cycles with
molecular oxygen and producing active oxygen species
and oxidative stress2I Quinlones being widely distributed
in nature and in clinically important drugs undergo fla-
voprotein-catalyzed redox cycling leading to the produc-
tion of reduced oxygen species which ultimately cause
cell damage:.22In the preseince of adequate Vit K3con-
centrations, hydrogen peroxide formation has been dem-
onstrated in ascites cells.6
The resullts of combined administration of Vit C and
K3 may well fit in with the hypothesis of hydrogen per-
oxide (H20;) formation by both of these vitamim6 Indeed
the cytotoxic:actions of quinone anti-cancer drugs appear
to be mediated by their free radical metabolites. Analyzing
the ascorbate-quinone interactions on Ehrlich ascites tu-
mor cells in vivo, a direct correlation between the cyto-
toxicity of the quinone/ascorbate preparation and the
generation of long lived senniquinone free radicals is ap-
parent.23Furthermore, although quinones are mostly re-
duced enzymatically by flavoproteins, in a one-electron
or a two-electron transfer, intracellular reductants as as-
corbate are able to achieve the same effect.24These hy-
pothetical mechanisms appear to be confirmed by the fact
c AND K3 ACTION * Noto et d. 905
that the inhibitory effects of combined Vit C and K3 are
completely abolished by the simultaneous addition of
catalase to the growing cell lines. Indeed, the lethal effects
of ascorbate on Ehrlich ascites carcinoma cells in vitro
which are synergistically enhanced by 3-amino- 1,2,4-tria-
zole, is neutralized by catalase. In these circumstances the
stimulation of the ascorbic effect by amino triazole has
been ascribed to its inhibition of cellular catalase respon-
sible for the detoxification of hydrogen peroxide.6Another
contributory factor to the antineoplastic action of Vit C
and K3appears to be the reactivation of nucleases in tumor
cells which are deficient of these enzymes thus exposing
the integrity of their nucleic acids.25
The inflicted damage by combined Vit C and K3 treat-
ment to the different cell lines appears dramatic and ir-
reversible. Indeed the effects of a 1-hour exposure to com-
bined vitamin treatment remains noticeable at the time
control nontreated cell lines reach confluence. It is not
unlikely that the effects of Vit C and K3, apart from tox-
icity induced by H202formation and catalase reduction,
is further enhanced by a number of additional factors
~ ascribed to each of these compounds individ-
ually, contributing to achieve synergism. These observed
in vitro synergistic effects confirm the in vivo tumor growth
inhibition by combined Vit C and K3 potentiating che-
motherapy. I
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