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-mentioned effects.

Cancer 63:901-906, 1989.

MONG CHEMICAL co:mpounds attracting great in- A 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/ 1 OTYz 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 K3 inhibits 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~.’~’’~ In vivo administration of vitamins C and K3 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 CANCER Ma? *ch I 1989 Vol. 63

I T

Y v) m - - 150 L W n + c W + 6 100 "

T

2 4 6 0 12 16 20 D A Y S

FIG. 1. Growth curve of MCF-7, KB and AN3-CA cell lines. Approx- imately 1 X lo6 MCF-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)$304 was 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. lo6 per 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), 1 TO nonessential 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 ethylenediamine tetraacetic 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.

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/

x 100 flask of treated cells

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 OF 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 l o3 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

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-CA cell lines required for a similar effect, levels in excess of respectively 5. 104 nmol/ 1 (Fig. 3) and lo4 nmol/l (2.76 pglml) concentrations (Fig. 4).

FIG. 3. Inhibition of KB cell growth by separate exposure to dif- ferent concentrations of vitamin C or K,, or combined both vitamins C and Ks. Techniques and symbols as in Figure 2

VI - T 1 0 0 -

m

L 80-

E 6 0 - n

u -

Y

0

c 0

-

._ c t - .-

LO- f

b 2 0 -

- 0

Efects of Combined Vitamins C and K3

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 5.103 nmol/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 1 O5 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

- - 1 10 10’ 2.10‘ 5.10’ lo3 5.10, 10‘ Concentration o f VIT C (pMo la r )

10 lo2 lo3 2.103 5.10, 10‘ 5.10‘ 10’ Concentration o f VIT K, (nMolar)

s

904 CANCER March I 1989 Vol. 63

1 10 l o2 2 lo2 5.102 lo3 5.103 10' Concentrat ion o f VIT C (pMo la r )

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

nmol/l (1.38 pg/ml) practically no inhibition was observed on the KB cell line. When, however, both vitamins were used in combination at the same concentrations an almost 50% inhibition was observed (Fig. 3). At Vit C levels of lo3 pmolll and Vit K3 levels of lo4 nmol/l used separately, no in vitro cytotoxic effects were seen on KB cell line, whereas when these two vitamins were applied in com- bination at the same concentrations, the percentage of inhibition reached 100%.

On the AN3-CA cell line Vit C and Vit K3 administered separately, at concentrations of 200 pmol/l (39.6 pg/ml) and 2. lo3 nmol/l (0.55 pg/ml), respectively, were practi- cally ineffective, whereas used in combination at the same concentrations an inhibition of 36% was observed (Fig. 4). A cytotoxicity of about 80% was reached when Vit C and Vit K3 were used in combination at concentrations of 500 pmol/l and 5. lo3 nmol/l, respectively.

+ .- .- LO[

/

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.

Efect of Catalase

At a concentration of 50 pg/ml catalase added to grow- ing AN3-CA cells did not affect the growth properties of this cell line. However, when AN3-CA cells were cultivated as described and simultaneously incubated for 1 hour with catalase (50 pg/ml; 367.5 U/ml) and increasing levels of combined Vit C and K3, the previously observed growth inhibitory effects of Vit C and K3 assessed upon confluence of the control nontreated cell line had completely disap- peared even at the highest vitamin concentrations ex- amined (Fig. 5).

Discussion

DNA determinations closely reflect the number of cells undergoing growth inhibition either due to an inhibition of cell division or/and cell death." It is shown that Vit C

s m 2oz lo2 2.102 5.102 lo3 5.10' 10' Concentration o f VIT C (pMo la r )

103 2.10' 5.10' 10' 5.10' lo5 Concentration o f VIT K, (nMolar)

FIG. 5. Counteraction of catalase (added at the dose of 50 pg/ml) against the AN3-CA cell growth in- hibitory effect of combined vitamins C and K3 (0). AN3-CA cell growth inhibition pattern was obtained by combined vitamins C and K3 treat- ment (0). Techniques as in preceed- ing figures.

No. 5 SYNERGISM OF COMBINED VIT c AND K3 ACTION * Noto et d. 905

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 a n ~ t h e r . ~ . ~ . ~ ~ . ' ~ Vitamin K3 when given alone, at below 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 . ~ . ~ . ~ ~

The most established function of Vit K3 is its role in regulating the synthesis of blood coagulation factors. '*,I9 However, Vit K3 may also inhibit the binding of the epi- dermal growth factor (EGF ) to the membrane receptors, cause membrane-lipid rearrangement and alter calcium fluxes. 12,20 The 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 stress2 I 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:.22 In the preseince of adequate Vit K3 con- 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.23 Furthermore, 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.24 These hy- pothetical mechanisms appear to be confirmed by the fact

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.6 Another contributory factor to the antineoplastic action of Vit C and K3 appears 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 H202 formation and catalase reduction, is further enhanced by a number of additional factors specifically 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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