PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS

Interrelationship of Ascorhate, Arachidonic Acid and Prostaglandin E2 in B16 Melanoma Cells

K. E. Stoll. P. Ottino and J. R. Duncan

Department oj’Biochemistr_y and Microbiology. Rhodes University, PO Bos 94. GrahamstoHvl. 6130. South tlfricu (Reprint requests to KES)

ABSTRACT. Previous studies have shown that ascorhate (Asc) supplementation affects arachidonic acid (AA) and prostaglandin Ez (PGEJ levels in B16 murine melanoma cells. In this study, non-malignant LLCMK cells and malignant B16 cells were respectively supplemented with 20 PCi 15-3H AA, to investigate whether these two cell types were able to take up AA from the media. Furthermore, these cells were also supplemented with Asc (O-100 pg/ml) to determine the effect of Asc supplementation on 15-3H AA uptake. Both cell types incorporated 15-3H AA, while Asc supplementation enhanced this 15-3H AA uptake. To determine the site of the AA incorporation, both cell types were supplemented with 2.5 FM AA and Asc (O-100 pg/ml). The % AA composition of the stroma fractions of both cell types was increased with 100 pg/ml Asc supplementation. Supplementation of these cells with AA (O-50 FM) resulted in an increase in PGEz levels in the B16 cells. Since PGE,! has been shown, in turn, to stimulate adenylate cyclase (AC) activity, the LLCMK and B16 cells were supplemented with O-100 pM PGE2. A 3-fold increase of AC activity in the B16 cells occurred as a result of this supplementation.

INTRODUCTION

Mammals have to obtain arachidonic acid (AA) from their diet or synthesize it from linoleic acid (LA) by

means of an elongation and desaturation process (1, 2). The free AA, in turn. is required for action by cylooxy- genase and lipoxygenase. thus becoming a major factor

in the regulation of eicosanoid synthesis (3-5). Both AA and the prostaglandins (PGs), PGE, in particular, play a key role in cellular regulation (6). They are thus able to

influence the growth and metastasis of tumours (7). PGs are cell-to-cell messengers in both non-malignant and

malignant cells (8-10) and are not stored in the cell to any great extent (8. 11). Hence, stimulation of their release requires the prior mobilization of substrate

precursors (8, 11). .4s most AA is esterified to specific phospholipids,

e.g. phosphatidylcholine. cells usually contain small

amounts of free AA (2). Fatty acid (FA) supplementa- lion of cells in vitro or changes in dietary fat can lead to marked changes in the FA composition of membrane

phospholipids ( 12, 13). These modifications are exten- sive enough to alter membrane fluidity, as well as affect

a number of cellular functions such as cell growth (14). Tumour cells are reported to have altered membrane

113

fluidity due to changes in the membrane polyunsaturated FA (PUFA) content (15). The influences that FAs exert

on the fluidity and/or order of a membrane have wide- spread implications for the functioning of those proteins

whose actions depend on mobility within the plane of the membrane ( 12).

A major intracellular signalling system identified so

far is that of adenylate cyclase (AC), which generates CAMP from adenosine triphosphate (ATP) and is regu- lated be guanine nucleotide regulatory proteins ( 1, 16).

The action exerted by PGs in many biological systems has been shown to be associated with the activation

of AC (9, 1 1, 17). Changes to the membrane intracellular

signalling systems are frequently implicated in the development of neoplasia ( 16). As the transformation of cells leads to a dramatic change in the lipid structural organization within the tumour cell membrane (18. 19)

and the activity of many integral membrane-bound enzymes is modulated by the nature of its membrane lipid environment (20, 31). this can affect AC activity.

Ascorbate (Asc), a reducing/oxidizing agent, affects

lipid metabolism (22) and is known to be essential for the structural integrity of the intracellular matrix (23). Asc supplementation at pharmacological levels to cells in culture, has been shown to exert either a stimulatory or inhibitory effect on the growth of these cells (24-28). In human platelets (29) Asc supplementation had no effect on the conversion of AA to PGE,. However. it was

124 Prostaglandins Leukotrienes and Essential Fatty Acids

found that the release of PC& in human parenchyma was promoted with Asc supplementation (30).

This study was undertaken to investigated the effect of Asc, AA and 15-3H AA supplementation on the

growth of non-malignant LLCMK and malignant B16

cells. The effect of Asc supplementation on the uptake of

15-3H AA into the cells was also investigated, together

with the effect of Asc supplementation on the incorpora- tion of AA into the membrane and stroma fractions of

the two cell types. Previously, we reported an increase in AA content and hence PGEz levels in B16 cells with

decreased cell growth, upon Asc supplementation (31). Therefore, another aspect investigated here, was the effect

of AA supplementation on PGE, levels in the LLCMK

and B16 cells, in the absence of Asc supplementation.

Further investigation involved the analysis of AC activity

in these two cell types, with only PGE, supplementation.

MATERIALS AND METHODS

Materials

Non-malignant LLCMK (monkey kidney) cells and

malignant B16 murine melanoma cells were obtained

from the Highveld Biological Association, South Africa. A SP 2330 GLC column was manufactured by Supelco

and supplied by Anatech Instruments, South Africa. Methylated FA standards were purchased from Nu Chek

Prep, Inc., Minnesota, USA. Ready-Solv’” EP scintilla- tion cocktail was purchased from Beckman, Ireland. AA

was obtained from Sigma Chemical Co., USA and 15-3H AA from Amersham International, England. SEP-PAK

C,s Cartridges were purchased from Waters Associated, Inc., Massachusetts, USA. PGE2 [125I] assay kit with Amerlex-M” magnetic separation was obtained from Amersham International, UK. Methyl formate, aluminum oxide 90, creatine-P-Na, 4H20 and creatine kinase were

purchased from Merck, Darmstadt, Germany. 3-Iso-

butyl- 1-methylxanthine, CAMP H20 and ATP-Na2H3 3H20 were obtained from Sigma Chemical Co., USA.

EDTA disodium salt was purchased from Holpro Chemical Corp., South Africa.

Cell culture

(A) 3 x lo5 non-malignant LLCMK or malignant B16 murine melanoma cells were respectively seeded into 6 sets of 5 25 cm2 flasks. 10 ml of MEM Basal Media,

containing 10% foetal calf serum (FCS), was added to all the flasks. Asc, over the concentration range 25-100 mg/ml, was added to 4 sets of the flasks. The 6 sets of flasks were incubated at 37°C with one media change. 18 h prior to confluency, 20 pCi AA was added to 5 sets of the flasks and the flasks incubated again. The sixth set was thus the control set, i.e. no Asc or 15-3H AA supplementation.

(B) The above methodology (A) was repeated with the following changes. 5 x lo5 non-malignant LLCMK

or malignant B16 murine melanoma cells were respec-

tively seeded into 6 sets of 5 75 cm* flasks. 30 ml of MEM Basal Media, containing 10% FCS, was added to

all the flasks. Once again, Asc supplementation over the

concentration range 25-100 pglml, was performed on 4

sets of the flasks. 18 h prior to confluency, 5 sets of flasks were supplemented with 2.5 pM AA and the flasks

incubated again. The control set was again the sixth set

of flasks, and contained no Asc or AA supplementation. (C) 5 x lo5 non-malignant LLCMK or malignant B 16

murine melanoma cells were respectively seeded into 75 cm’ flasks. 30 ml of MEM Basal Media, containing 10% FCS, was added to all the flasks. The flasks were

incubated at 37°C with one media change. 24 h prior to

contluency, 1 pM AA, 5 pM AA, 10 pM AA and 50 pM

AA was added to the flasks and the flasks incubated again. 5 duplicates were set up at each concentration, as

well as a control set of flasks.

(D) The above methodology (C) was repeated with the following changes. 3 x lo5 non-malignant LLCMK or

malignant B 16 murine melanoma cells were respectively seeded into 25 cm* flasks. 10 ml of MEM Basal Media,

containing 10% FCS, was added to all the flasks. Once again, the flasks were incubated at 37°C with one media change. 24 h prior to confluency, 1 pM PGE*, 5 pM

PGE?, 10 pM PGE? and 100 pM PGE, was added to the flasks and the flasks incubated again. 5 duplicates were set

up at each concentration, as well as a control set of flasks. Upon confluency, cells in every flask in all the experi-

ments were harvested using 10 ml of trypsin solution (500 units/ml). These cell suspensions were centrifuged

at 3000 g for 10 mitt, and the pellet resuspended in 1 ml phosphate buffered saline (PBS) solution. The cells were counted using a haemocytometer.

Homogenisation of the cells and separation of the cellular components

The respective cell suspensions were poured into a

Dounce homogenizer and homogenized 30 times with the tight plunger. The various homogenates were then centrifuged at 480 g for 20 min, to remove nuclei and non-disrupted cells. The supematant was retained and

centrifuged at 4000 g for 20 min, to remove the mito- chondrial fraction. Once again, the supematant was retained and centrifuged at 20 000 g for 30 mitt, in order to obtain the respective stroma (supematant) and membrane (pellet) fractions of the homogenized cells. The pellet

was resuspended in 1 ml PBS.

Determination of 1S3H AA uptake

The respective 1 ml stroma and membrane fractions of the two cell types supplemented with 15-3H AA, were placed into scintillation vials which contained 5 ml of scintillation cocktail. The amount of 15-3H AA in each fraction was determined using the Beckman LS 3801 scintillation counter.

Interrelationshio of Ascorbate. Arachidonic Acid and Prostaylandin E, in B 16 Melanoma Cells 135

Saponification, esteritication and extraction of FAs

The method used was that of Skeef (32). The various cell fractions of (B) were transferred into round-bottom flasks and 2 ml of 10% methanolic KOH was added to

each flask. The lipids were saponified by heating with reflux under nitrogen for 45 min at 85°C. Upon the completion of saponification, they were acidified by

adding 1 ml of 7 N HCl in order to free the FAs. The

FAs were extracted twice with 3 ml of petroleum ether and vortexed for 2 min each time. The petroleum ether

extracts were pooled, and evaporated to dryness under nitrogen at 60°C. The residual FAs were methylated by

heating with 0.3 ml BF,-methanol reagent, with reflux

and under nitrogen for 5 min at 100°C. The FA esters

were again extracted, twice, using 1 ml petroleum ether,

as well as vigorous shaking for 2 min each time. The pooled extracts were evaporated to dryness under nitrogen

and reconstituted with 20 pl petroleum ether. They were

stored at -20°C under nitrogen and protected from light until analysed by gas-liquid chromatography (GLC).

Free FA anatysis by GLC

GLC. with a SP 2330 fused capillary column in the

Hewlett-Packard 5890 GLC, was used to obtain the separation of the free FAs which were reconstituted in

the petroleum ether. The temperature program involved an initial heating time of the column at 130°C for 15 min,

followed by an increase in temperature of 4”Clmin to obtain 22O”C, at which temperature the column was

retained for the final 7.5 min of the run. The sample volume of reconstituted FAs, loaded onto the SP 2330 column, was

1 ~1. AA (20:4) levels were expressed as % composition of 4A relative to the total amount of FA in that fraction.

Extraction and isolation of PGs

The PG’s were extracted according to a modified

method of Powell (33). The respective 1 ml cellular frac-

tions were added to 15 ml cold ethanol (95%) and

shaken for 10 min. The sample was diluted to 100 ml with cold water and vortexed for 1 min. The pH of the suspension was adjusted to 3.0 using 1 N HCl, and the hample then passed through a SEP-PAK cartridge, which

had previously been wet by passing 20 ml of 80% aque- ous ethanol through it and then washed with 20 ml water to remove excess ethanol. Before the elution of the PGs

using 5 ml methyl formate, the cartridges were first washed

with 10 ml water and 10 ml petroleum ether. The eluant was dried under a stream of nitrogen at 25°C. The SEP-

PAK cartridges were regenerated for reuse by washing with 30 ml of 80% ethanol followed by 20 ml water.

PGEz [lz51] assay system

The extracted, dried PG fraction was reconstituted with 100 FM of assay buffer, (pH 7.0). 100 l_tl methyl oximation reagent was added to the reconstituted sample

and vortex mixed. The resultant solution was incubated

at 60°C for 1 h, to allow methyl oximation of the sample

to occur. The preparation of the samples for scintillation counting, using the Beckman Gamma 3 10 Scintillation counter, was as described in the assay kit.

Protein determination

The protein concentration of the stroma and membrane fractions was determined by the method of Lowry et al

(34).

AC assay

AC activity of the membrane fraction was determined

using the method of Schultz and Jakobs (35). and meas-

ured the rate of the conversion of [A-“P] ATP to [j’P]

CAMP. No guanosine triphosphate (GTP) or forskolin was added to the reaction mixture, instead 30 ~1 of

TRIS-HCL buffer was substituted in order to give the desired final volume of the mixture. Time course assays were performed over a period of 10 min. Optimal AC

activity was detected after a 45 s incubation period of

membrane homogenate and substrate solution. AC specific activity was calculated as catalytic activity relative to

the mass of protein, expressed as the formation of

1 pmol CAMP per min and mg protein.

Statistical analysis

The results obtained were analyzed using a one way analysis of variance (ANOVA) followed by the Student- Newman Keuls Multiple Range Test.

RESULTS

The effect of Asc and AA supplementation on the growth of the cells

Asc and AA supplementation (radioactive and non-

radioactive) resulted in an overall decrease in the cell growth of both the LLCMK and B16 cells (Table 1 and

Table 2). The decrease in LLCMK cell growth. at 25 pg/ml

Asc and 15-jH AA supplementation was significant (p < 0.05) (Table 1). Prior to Asc supplementation, AA

and 15-jH AA supplementation did not affect B16 cell

growth to any extent. However, at 100 pg/ml Asc and 15-“H AA supplementation, the decrease in B 16 cell

growth was significant (p < 0.00 1) (Table 1). Although not significant, B 16 cell growth decreases markedly with Asc and 2.5 PM AA supplementation.

The effect of Asc supplementation on 15-3H AA uptake by the cells

The uptake of 15-‘H AA in the LLCMK stroma fractions was greater than the uptake of 15-“H AA in the mem-

126 Prostaglandins Leukotrienes and Essential Fatty Acids

Table 1 The effect of Asc and 15-3H AA supplementation on the Table 2 The effect of Ax and AA supplementation on the growth of growth of the LLCMK and B16 cells, respectively. Results are the LLCMK and B16 cells, respectively. The results are the mean of five mean of five cultures f SE cultures f SE

Asc 15-3H AA Cell No. x 10a/ml

Wml @i LLCMK B16

0 0 36.0 362.9 f 5.5 k 22.4

0 20 28.8 368.6 k 3.2 + 12.9

25 20 14.3h 384.3 i 0.9 f 40.3

35 20 29.1 349.2 * 4.5 f 34.2

50 20 26.0 394.1 f 1.8 I? 10.9

100 20 27.2 181.0” * 2.2 * 35.4

a = p I 0.001: relative to the control and other B 16 cultures supplemented with 15-3H AA. b = 20.05: relative to the control and other LLCMK cultures supplemented with 15.-‘H AA.

Asc [AAl @ml FM

0 0

0 2.5

25 2.5

35 2.5

50 2.5

100 2.5

Cell No. x lW/ml LLCMK B16

365.7 823.0 +I 7.8 k 25.6

354.3 805.0 rt 14.1 f 91.2

388.5 631.0 f 11.4 * 7.0

357.3 619.3 f 22.5 f 69.9

368.8 728.3 * 2.6 f 41.9

336.3 767.7 f 1.7 z!c 32.9

brane fractions. A decrease in 1S3H AA uptake in these membrane fractions occurred with increasing Asc

supplementation. The level of 1.5-“H AA in the LLCMK stroma fraction

without Asc supplementation, was significantly lower (p 5 0.05) than the amount of 1S3H AA detected in

those stroma fractions supplemented with 25 and 50 pg/ ml Asc (Table 3). In contrast, the level of 15-3H AA in

the LLCMK membrane fraction not supplemented with Asc, was significantly higher (p < 0.025) than the amount of 15-3H AA detected in the membrane fractions

supplemented with 35,50 and 100 pg/ml Asc (Table 3).

detected in the 100 pg/ml Asc and 15-3H AA supple-

mented B 16 stroma fraction was significantly (p I 0.05) higher than the 15-3H AA detected in other Asc supple-

mented stroma fractions (Table 3). The level of 15-3H AA in this stroma fraction was now greater than the

15-3H AA detected in the corresponding membrane frac-

tion. This latter effect was also found for the stroma and membrane fractions of the B 16 cells supplemented with

50 pg/ml Asc and 15-3H AA. The level of 15-3H AA detected in the 0 and 25 pg/ml Asc supplemented B16 membrane fractions was significantly higher (p IO.005

and p 50.01, respectively) than the 15-3H AA found in the 35, 50 and 100 pg/ml Asc supplemented membrane

fractions. A comparison of the B 16 stroma and membrane frac-

tions supplemented only with 15-3H AA, revealed that

the level of 15-3H AA in the membrane fraction was greater than that in the stroma fraction. The 15-3H AA

In the B16 cells, the significant decrease in cell growth

at 100 pg/ml Asc and 15-3H AA supplementation corre- sponded to a significant increase in the level of 15-“H

AA in the stroma fraction of these cells. In general, as

Table 3 The effect of Asc supplementation on the uptake of 15.‘H AA by the LLCMK and B 16 cells, respectively. Values recorded are the mean of five cultures f SE

[A=1 15-3H AA 1 5-3H AA in stroma 15-3H AA in membrane

Wml pCi c.p.m./104 cells c.p.m./104 cells LLCMK B16 LLCMK B16

0 0 0 0 0 0

0 20 264.2a 324.3 185.0b 45 1.9* * 59.8 k 20.4 f 67.4 k 26.4

25 20 558.2 247.1 94.5 372.6d

k 14.4 k 42.8 f 22.1 * 41.4

35 20 309.3 162.1 33.5 190.1 f 32.8 k 40.3 + 5.2 * 77.4

50 20 460.5 171.4 41.3 93.6 f 48.0 * 11.9 k 3.8 f 10.5

100 20 253.9’ 465.8’ 12.8 184.4 Yk 45.7 f 103.4 * 1.0 k 23.5

a = p < 0.05: relative to the 25 and 50 Kg/ml Asc supplemented LLCMK stroma fractions. b = p < 0.025: relative to the 35,50 and 100 pg/ml Asc supplemented LLCMK membrane fractions. c = p IO.05: relative to the 25, 35 and 50 &ml Asc supplemented B16 stroma fractions. d = p < 0.01: relative to the 35, 50 and 100 yg/ml Asc supplemented B16 membrane fractions.

Interrelationship of Ascorbate. Arachidomc Acid and Prostaglandin E, in B I h hlelsnoma Cell\ I27

B16 cell No x ld?ml 6oo, 15% AA uptake (cpm/l~cells\ ,ooo !

400 800 ~

300 \ , 600 ‘\

200 c ‘1

‘.. ‘k j;x 400

‘._ ---i

loot ,,.i j 200

0 1__ I I 1

Control 0’ 25’ 35’ 50’ 10

100.

IAscorbatel pg/ml

+ B16 growth -s+ AA uptake l = POuCi

Fig. I The effect of Axe woplementation on the total uplahc 01 I S-‘H /IA by the

B16 cells. .

B I o cell growth decreased, so the overall level of 15’H

AA increased. The opposite effect was also observed, i.e. an increase in B 16 cell growth was accompanied by

a decrease in I S3H AA content (Fig. 1).

The effect of Asc and AA supplementation on the % composition of AA in the cells

The 5% AA composition in the LLCMK stroma fractions

of rhe control, the 3.5 PM AA supplemented, and the SO pg/ml Asc and 2.5 yM AA supplemented cells was significantly higher (p I 0.0 I: p I 0.05 and p I 0.01. respectively) than the % AA in the other stroma fractions (Table 4). The % AA composition in the LLCMK control

stroma fraction was also higher than that detected in the corresponding membrane fraction.

This latter observation was also made concerning the control stroma and membrane fractions of the B 16 cells.

Generally. however, the 5% AA composition in the B 16 membrane fractions was higher than that in the stroma

fractions. Upon 25 pg/ml Asc and 2.5 yM AA supple- mentation. the 5% AA composition in the B 16 membrane

fraction increased significantly (p IO.05). Increasing Ax supplementation resulted in a decrease in membrane 9; AA composition of these cells.

In Figure 2, it can be seen that upon Asc and AA

supplementation where B16 cell growth decreased, the C/C AA composition increased. The opposite effect also

holds true, i.e. as the growth of these cells increased so the % AA composition was reduced.

The effect of AA and PGE, supplementation on cell growth

AA and PGE, supplementation of the LLCMK cells resulted in a trend of slightly decreased cell growth (Table 5 and Table 6). The same effect was observed

Table 4 The effect of Ax and AA supplementatwn on the ‘/r .AA

composition in the respective htroma and membrane iraction\ 01 the

LLCMK and B 16 cells. Results are the mean of fire cuIturc\ + SE

[Ax] [AA] ‘*AA in stroma f/i 4A In membrane

&/ml PM LLCMK Blh LI.CMK HI6

0 0 6.17,# ;.w 2.5x I.111

* I.17 + (I.(17 * 0.w + o..w

0 -Ii 5.w (I.X-l 1.77 I.57 i 0.2.i i 0.01 4 I).-~(1 * 0.56

25 2.5 I.61 I1.W 5.52 S..<>’

+ O.hI * II.0’) z i.lK t 0.114

35 2.5 O.Xh 7.11 1. io :.51 * I).IX t Il.21 ‘I II. !‘I -I 0.w

50 1.5 (1.1 T I .7(> 4.1-1 L..iS * O.hfl + (I.18 i (1.S’ f (1.3h

100 1.5 I .‘I7 ?.I,? hi5 I.OI f 0.7’) -f- 0.10 f 2.15 f (1.1’)

--

a = p 5 0.0 I: relative to the 75. 35 and IO0 ps/ml AIC wpplcmcntcd

LLCMK woma fractions. h = p 5 0.05: relative to ihc 75. i5 and

100 pg/ml A\c wpplcmentcd LLCMK womn Iraction\. c = p 5 0.01:

relative to the 75. 35 and IO0 @ml A\c supplemented L.LCMK stroma fraction\. d = p 5 0.05: rclntivc to all the H IO mcmhl-ant

fractions. except for that at 35 mg/ml Aw \Llpplemst~lati[)n.

with AA supplementation of the Bl6 cells (Table 5). However. PGE, supplementation of the 3 I6 cells resulted initially in a significant (p 2 0.00 I ) increase in cell growth

at I /_tM PGE, supplementation. Cell growth was subse- quently reduced with increasing PGE, supplementation and

at 100 PM PGE, supplementation there was a significant (p 5 0.001) decrease in B 16 cell growth.

The effect of AA supplementation on the PGE, levels within the cells

In both the LLCMK cells and the B 16 cell<. the levels of PGE, in the stroma fractions were significantly greatel

I28 Prostaglandins Leukotrienes and Essential Fatty Acids

El6 cell No x lO?ml Total % AA composition

[Ascorbatel pg/ml

+- Bl6 growth -% AA l n 2.5 UM

Fig. 2 The effect of Asc and AA supplementation on % AA composition in B16 cells

Table 5 The effect of AA supplementation on the growth of the Table 6 The effect of PGEz supplementation on the growth of the

LLCMK and B 16 cells, respectively. Results are the mean of five LLCMK and B 16 cells. respectively. Results are the mean of five

cultures f SE cultures f SE

lAA1

W

0

1

5

10

50

Cell No. x IF/ml LLCMK Bl6

614.2 916.8 k 64.8 + 119.2

635.1 699.3 f 46.1 * 19.9

556.5 725.9 * 54.3 + 44.6

554.7 809.6 * 39.1 k 48.6

641.4 163.4 + 48.0 k 58.3

(p 5 0.005) than the PGEz levels in the respective mem- brane fractions (Table 7). However, PGEl levels in the B 16 cells, in both fractions, were greater the those of the LLCMK cells.

IPGEJ Cell No. x lo-‘/ml

!JM LLCMK B16

0 168.4 171.1 + 16.5 * 10.0

I 1.58.4 226.0” k 21.8 i- 9.4

5 155.2 147.0 k 8.7 * 7.6

IO 144.8 137.0 + 12.5 k 4.9

100 147.8 60.0h f 11.1 * 0.0

a = p 2 0.00 1: relative to the B I6 control cells and the other PGE, supplemented B 16 cells. b = p 2 0.001: relative to the B 16 control cells and other PGEl supplemented cells.

The effect of PGE2 supplementation on AC activity in the cells

PGE?, detected in the control stroma fraction of the LLCMK cells, was lower than the PGEz found in the

stroma fractions of AA supplemented LLCMK cells. At 5 pM AA supplementation, the levels of PGE? increased

significantly (p < 0.05). In the LLCMK membrane fractions, the PGEl levels in both the control and AA

supplemented cells were similar (Table 7).

AC activity detected in both the control and PGE-, supplemented LLCMK cells was significantly higher

(p IO.005) than that of the B 16 control cells (Table 8).

The levels of PGE? in the B 16 stroma fraction supple- mented with 1 pM AA decreased relative to that of the control stroma fraction. Further AA supplementation resulted in an increase in PGE, levels, especially in the B 16 stroma fraction supplemented with 50 pM AA (Table 7). An increase ‘in PGE, was found in the membrane fraction of B 16 cells supplemented with 1 pM and 50 pM AA. This increase was significant (p I 0.05) in the 50 yM AA supplemented B 16 cells.

PGE, supplementation did not appear to affect the AC activity in the LLCMK cells to any extent. However,

with regard to the B16 cells, AC activity in the control

cells was lower than that of the PGE, supplemented cells, although not significantly so.

DISCUSSION

Asc supplementation has been reported to have both an inhibitory and a stimulatory effect on the growth of tumour cells (24-27). In this study, the effect of Asc supplementation (over a nutritional range) and AA

Interrelationshio of Ascorbate. Arachidonic Acid and Prostazlandin E, in B 16 Melanoma Cells 129

Table 7 The effect of AA supplementation on the PGE, levels in the stroma and membrane fractions of the LLCMK and B 16 cells, respectively. Results are the mean of five cultures + SE

IAAI PGE: (pg/tube) PGE, (pg/tube)

W in stroma in membrane LLCMK B16 LLCMK B16

IJ 12.04 141.80 3.55 5.80 + 2.00 * 18.20 f 1.30 * 1.27

I 16.66 98.42 2.48 11.79 rf- 2.11 + ‘4.19 f 0.06 k 3.86

5 45.82,’ 137.20 5.71 4.57 i 9.70 k 19.27 -t 1.26 * 1.44

IO 10.38 124.80 4.04 2.70 k 0.69 k 18.71 k 1.07 f 0.44

50 17.36 160.00 3.79 14.22h Yk 2.63 f 0.00 f 0.85 k 1.87

a = p < 0.05: relative to PGE, levels detected in any of the other LLCMK stroma fracti0ns.b = p < 0.05: relative to PGE, levels detested in the 816 control. ~FM AA and 10kM AA supplemented membrane fractions.

Table 8 The effect of PGE? supplementation on the AC activity in the membrane fractions of the LLCMK and B16 cells, respectively. Results are the mean of five cultures f SE

[ PGE?] @I

I)

AC activity (U/mg) LLCMK cells B 16 cells

3.34 0.59”

* 0.5 1 + 0.07

I 3.78 1.47

k 0.64 f 0.30

5 3.09 1.94

k 0.51 + 0.67

10 3.28 1.69

i- 0.42 * 0.30

IO0 3.06 1.69

k 0.57 k 0.31

a = p 5 0.005: relative to AC activity in the control and PGEz supplemented LLCMK cells.

supplementation (including 15”H AA) was that of a general decrease in the growth of both the LLCMK and

B 16 cells. The only significant decrease in cell growth was observed in the B16 cells supplemented with 100 yglml Asc. Thus, the inhibitory effect on cell

growth, when supplementing with Asc over a nutritional range, is not as dramatic as when Asc supplementation occurs over a pharmacological range (24, 26). However,

as Asc supplementation over a pharmacological range is also reported to be stimulatory to tumour growth (25), it therefore appears that the effect of Asc supplementation on tumour cells could be dependent on the cell type.

Certain FAs in high concentrations can lower the growth rate of cells without killing cells, while others

report that some FAs may exert selective toxicity against

neoplastic cells (36). Human skin fibroblast growth was reduced by 25-50% when palmitate. linolenate or AA

was added in concentrations of 50 PM or above ( 14). Human breast, lung and prostate cancer cells are

reportedly selectively killed when supplemented with

n-6 EFAs (37). It was found that AA supplementation resulted in a general decrease in LLCMK and B16 cell growth, in this study. Although not significant, this

effect was greater in the B 16 cells. When supplementing

with PGE,, 100 pM PGE, significantly decreased B 16 cell growth. Another study (38) done on the B 16 murine

melanoma model, also shows eicosanoid synthesis to be negatively correlated with metastatic potential. It was

found that PGE, concentrations below I x 10.’ M did not

affect the proliferation rate of murine mammary tumour

cells (39). The growth of the human gastric carcinoma

cell line, KATO III, was also inhibited by PGE, supple-

mentation (40). PGE? increased the growth of Raji lym- phoid cells at lower concentrations. while at higher concentrations PGE? inhibited the cell growth signifi-

cantly (41). In this study, PGEz supplementation did not

affect LLCMK cell growth to any extent. The uptake of 15-jH AA by the LLCMK and B16

cells. indicates that AA is able to permeate these cells.

However, the mechanism of PUFA permeation into cells

is still unknown. The AA entry step appears to be insen- sitive to the intracellular metabolism of the FA and is

therefore not driven by transmembrane concentration

gradients (42, 43). Present evidence is that a common entry path exists for AA and other long-chain free F.4s.

and the entry is mediated by a mechanism of facilitated diffusion driven by transmembrane potential difference

(43). AA unidirectional influx exhibits the features of a saturable process (42. 43). Asc also has limited cell membrane permeability, which means that the transport must be facilitated (44).

Significantly less 15-3H AA wab taken up into the LLCMK membrane fractions supplemented with

increasing Asc. The uptake of 15-“H AA by the B 16

membrane fraction in the absence of Asc. as well as in

the presence of the lowest concentration of Asc. was sig- nificantly greater than that in the membrane fraction at

higher concentrations of Asc supplementation. Upon Asc supplementation, the 15-‘H AA content of the B16 membrane fraction decreased relative to the 15-jH AA levels in the stroma fraction. At 100 pg/ml Asc supple- mentation the increase in 1 5-3H AA in the B 16 stroma fraction was in fact significant relative to that found at

lower levels of Asc. Thus, upon Asc supplementation there is a reduced uptake of 15-jH AA by the B 16 mem- brane accompanied by an increased uptake of the FA by

the stroma. The finding of significantly high % AA com- position in the membrane fraction at 2.5 pM AA and 25 pg/ml Asc supplementation supports this. At 100 pg/ ml Asc supplementation the % AA composition in the B16 stroma fraction was again higher than that of the membrane fraction.

130 Prostaglandins Leukotrienes and Essential Fatty Acids

An interesting observation was that the overall (com- bined stroma and membrane fraction) % AA composi-

tion in B16 cells, as well as the overall uptake of 1S3H

AA by the B16 cells varied inversely with B16 cell

growth. This supports an earlier study (31) where B16 cell growth was inversely related to the AA levels in the

cells, the latter supplemented with Asc but not AA. This

same study (31) also reported on the inverse relationship exhibited between B16 cell growth and PGE2 levels, together with the finding that the PGE2 levels in the B 16

membrane also decreased with Asc supplementation, while

that of the stroma increased with Asc supplementation.

Thus, the elevated levels of PGE2 in the B16 cells with

decreased growth upon Asc supplementation, may

be due to the elevated AA levels in the B 16 cells with

decreased growth upon Asc supplementation (31). It therefore appeared that Asc supplementation was not en-

hancing the conversion of AA to PGE,. The higher PGE? levels seemed the result of higher levels of the precursor, AA. This study, where B 16 and LLCMK cells were only

supplemented with AA, was aimed at verifying this. The

B16 cells supplemented only with AA, had increased total PGE? levels at 50 pM AA supplementation. PGE,

levels in the control B 16 cells were significantly higher than the PGE2 levels detected in the control LLCMK

cells. PGs, especially of the E series, have been shown to

be elevated in a large number of human and experimen-

tal tumours, and may play a role in the growth and spread of tumours (45,46).

Although changes to AC activity have been claimed not to be due to PUFA supplementation and the latter being

converted to PGs, AC activity is influenced by changes to the lipid environment, making it favourable for AC

activity (47). Supplemented FAs are incorporated into phospholipids in significant amounts and will therefore have an effect on membrane environment which in turn

affects AC activity (48). However, this does not rule out

the possibility that PGE, stimulates AC activity. PGE2 stimulated AC activity in the plasma membrane of KATO

III cells (49), and the same effect was observed in particulate fractions from rat liver (50). In this study, PGE, supple- mentation of the B 16 cells increased AC activity 3-fold. Optimum AC activity occurred at 5 pM PGE2 supple- mentation. This was a saturable process as further PGE,

supplementation did not appear to increase AC activity.

In conclusion, Asc supplementation generally enhances the uptake of AA by the stroma of the B16 cells. This AA has many an important function in the cells, one

being that the greater concentrations AA result in elevated PGE, levels in these cells. The higher PGE, levels, in turn, have various effects which include the stimulation of AC activity.

Acknowledgements

This work was partially funded by grants from the Foundation for Research and Development. National Cancer Association and Rhodes University.

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