Can tumour cell drug resistance be reversed by essential fatty acids and their metabolites?

Prostaglandins, Leukotdenesand Essential Fatty Acids (1998) 58(1), 39-54 © HarcourtBrace & Co Ltd 1998

Can tumour cell drug resistance be reversed by essential fatty acids and their metabolites? U. N. Das, N. Madhavi, G. Sravan Kumar, M. Padma, P. Sangeetha Division of Internal Medicine, Clinical Immunology and Biochemistry, L.V. Prasad Eye Institute, Road 2, Banjara Hills, Hyderabad-500 034, India

Summary Tumour cell drug resistance is a major problem in cancer chemotherapy. Essential fatty acids have been shown to be cytotoxic to a variety of tumour cells in vitro. But, the effect of these fatty acids on tumour cell drug resistance has not been well characterized. Gamma-linolenic acid (GLA) of the n-6 series and eicosapentaenoic acid (EPA) of the n-3 series potentiated the cytotoxicity of anti-cancer drugs: vincristine, cis-platinum and doxorubicin on human cervical carcinoma (HeLa) cells in vitro. Alpha-linolenic acid (ALA), GLA, EPA and docosahexaenoic acid (DHA) enhanced the uptake of vincristine by HeLa cells. In addition, DHA, EPA, GLA and DGLA were found to be cytotoxic to both vincristine-sensitive (KB-3-1) and -resistant (KB-ChR-8-5) human cervical carcinoma cells in vitro. Pre-incubation of vincristine-resistant cells with sub-optimal doses of fatty acids enhanced the cytotoxic action of vincristine. GLA, DGLA, AA, EPA and DHA enhanced the uptake and inhibited the efflux of vincristine and thus, augmented the intracellular concentration of the anti-cancer drug(s). Fatty acid analysis of KB-3-1 and KB-ChR-8-5 cells showed that the latter contained low amounts of ALA, GLA, 22:5 n-3 and DHA in comparison to the vincristinesensitive cells. The concentrations of GLA and DHA were increased 10-15 fold in the phospholipid, free fatty acid and ether lipid cellular lipid pools of GLA and DHA treated cells. These results coupled with the observation that various fatty acids can alter the activity of cell membrane bound enzymes such as sodium-potassium-ATPase and 5'nucleotidase, levels of various anti-oxidants, p53 expression and the concentrations of protein kinase C suggest that essential fatty acids and their metabolites can reverse tumour cell drug-resistance at least in vitro.

INTRODUCTION Efforts made to treat cancer with chemotherapeutic agents is often hampered by intrinsic or acquired drug resistance of the tumour cells. Some tumours initially respond favourably to chemotherapy, but subsequently develop muhi-drug resistance (MDR). Transmembrane transporter molecules, notably P-glycoprotein (PgP) or the multi-drug resistance associated protein (MRP) can mediate such drug resistance by acting as cytotoxic drug efflux pumps. In addition, multi-drug resistant tumour cells have been described without overexpression of either PgP or MDR indicating that other mechanisms may be operative. 1,2 One such mechanism that has been recently described include multi-drug resistant tumour cells overexpressing involves the 110-KD LRP (lung-resisReceived 27 January 1997 Accepted 3 April 1997 Correspondence to: Dr U. N. Das

tance-related protein)? LRP overexpression has been found to be associated with poor response to chemotherapy. It is now thought that cell cycle proteins called cyclins have a crucial role to play in controlling cell proliferation. There are about eight or so cyclins which are apparently oncongenes by themselves. The cyclins turn on enzymes called cyclin-dependent kinases (cdks) which can propel cells through the cell cycle (reviewed in 4). Cyclins and cdks contribute to the development of cancer, like oncogenes, by blocking cell differentiation and shortening the duration of cell cycle. However, there are also cell cycle inhibitors, which work like the negatively acting tumour suppressors, whose loss or inactivation leads to cancer. Three such inhibitors are p53, which blocks the activity of cdk2 and other cdks, transforming growth factor-beta (TGF-beta) and retinoblastoma (Rb) gene (reviewed in 4). In a recent study, Lowe et al~ showed that tumours expressing the p53 gene contained a high proportion of apoptic cells (apoptosis is defined as a process of cell death which has a genetic basis, in other words: geneti39

40

Das et al

cally programmed cell death) and typically regressed after treatment with gamma-radiation or adriamycin. On the other hand, p53 deficient tumours were found to be resistant to gamma-radiation or adriamycin and continued to grow and contained few apoptic cells. Acquired mutations in p53 were associated with both treatment resistance and relapse in p53-expressing tumours. These results 5 suggest that inactivation of p53 leads to defects in apoptosis, produces treatment-resistant tumours and suggest that p53 status may be an important determinant of tumour cell resistance or sensitivity to chemotherapeutic agents. It was noted that drug accumulation in muhi-drug resistant (MDR) cells can be regulated by protein kinases, in particular protein kinase C 0°KC)-mediated phosphorylation of pgp.6,7 In addition, several tumour cell lInes of the MDR phenotype exhibit increased PKC activity.a-l° Inhibition of PKC activity might therefore offer another avenue to counteract MDR. Though several drugs have been found to be effective in reversing MDR in vitro by virtue of their action on PgP, PKC and/or p53, none have so far found broad clinical application.1 ]-]3 Essential fatty acids (EF?,s) are precursors of eicosanoids and are important structural components of cell membranes (Scheme 1). They also provide the substrates for the generation of lipid peroxidation products which have an inhibitory action on cell proliferation? Tumour cells are known to have low delta-6-desaturase activity, an enzyme necessary for the desaturation of dietary linoleic acid (LA, 18:2n-6) and alpha-linolenic acid (ALA, 18:3n-3) to their respective products (Scheme 1).TM In an earlier study, we have shown that hepatocarcInogens, diethylnitrosamine (DEN) and 2-acetylaminofluorine (2AAF), can suppress the activity of deha-6-desaturase and

14

Concentration

///

40 /~g/ml

x

~8 ca P

~4 fi

,1:1 7

2

1

o •

3

1

2

•

3

t

2

3

in Days

Control • Docosahexaenoic acid v Eicosapentaenoic Gammalinc]enic acid [] D i h o m o g a m m a - l i n o l e n i c acid Linoleic acid zx A r a c h i d o n i c acid • Alpha-linolenic

acid acid

Fig. 1 Effect of various fatty acids on the survival of HeLa cells in vitro (cell number).

deha-5-desaturase resulting in low levels of gammalinolenic acid (GLA, 18:3n-6) and arachidonic acid (AA, 20:4n-6) in the tumour cells. 15,16These results led us and others to study the effect of various fatty acids on the survival of tumour cells in vitro. Addition of EFAs (LA and ALA) and their metabolites (such as GLA and AA of the n-6 series and EPA and DHA of n-3 series), to a variety of normal and tumour cells in vitro showed that only tumour ceUs are killed by these fatty acids without harming the n o r m a l cells. 17-2a This selective tumoricidal action of fatty acids seems to be mediated by free radicals and lipid peroxides. ]8-23 Similar to these fatty acids, radiation, some anti-cancer drugs and cytokines also seem to have the

°::::l ALA 1 8 : 3 n-3

GLA 18:3n-6 elongase

EPA 2 0 : 5 n-3 = P G s 1 series

D-5-D

AA 20 : 4 n-6

2

Time

D-6-D

DGLA 20 : 3 n-6

acid

12

LA 1 8 : 2 n-6

J J

of fatty

= P G s 2 series

I

DHA 22 : 6 n-3

Scheme I Showing the metabolism of Essential Fatty Acids Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 39-54

© Harcourt Brace & Co Ltd 1998

Can tumour cell drug resistance be reversed by essential fatty acids and their metabolites?

ability to generate free radicals in tumour cells and thus, may bring about their tumoricidal actions. 24-26 Since drug-resistance is a major obstacle in the clinical treatment of cancer and as EFAs and their metabolites (also called as cis-unsaturated fatty acids or c-UFAs) have selective tumoricidal action, studying the effects of cUFAs on drug-resistant tumour cells and their modulating influence on the actions of anti-cancer drugs has great clinical significance. Here, we present evidence that c-UFAs are toxic to vincristine-resistant and -sensitive (KB-ChR-8-5 and KB-3-1) h u m a n cervical carcinoma cells which are variants of HeLa cells, that c-UFAs can augment the cytotoxicity of anti-cancer drugs against HeLa cells, modulate the uptake and efflux of anti-cancer drugs by HeLa, KB-3-1, and KB-ChR-8-5 cells in vitro, and that the fatty acid composition of the phospholipid fraction of vincristine-sensitive and -resistant cells is quite different. In addition, the effect of c-UFAs on the activity of cell membrane bound enzymes: Na+-K+-ATPase and 5'-nucleotidase, PKC activity, anti-oxidant system of the tumour cells were studied and the results are reported here.

MATERIALS AND METHODS 1. Cells and culture conditions

The various types of cells that were used in the present study include:HeLa, KB-3-1, KB-ChR-8-5, SP 2/0 R and AK-5 cells. HeLa KB-3-1

: Human cervical carcinoma cells : a variant of h u m a n cervical carcinoma cells which are sensitive to the cytotoxic action of vincristine in vitro. KB-ChR-8-5 : a variant of KB-3-1 cells which are resistant to the cytotoxic action of vincristine in vitro. SP 2/0 Ag14 : a mouse myeloma cell line. AK-5 : a transplantable macrophageqike cell line. HeLa ceils were grown and maintained in NUNC culture flasks in bicarbonate-buffered RPMI medium containing 10% heat-inactivated h u m a n AB serum, 50 ~g/ml streptomycin and 50 units/ml penicillin. The h u m a n AB serum used was found to be free of hepatitis B virus and human immunodeficiency virus. Cells were seeded at I x 104 cells/ ml/well in 24-well tissue culture plates for viability and thymidine incorporation studies. The cells were grown in the medium at 37°C in a 5% CO 2 humidified incubator as described earlier. 22 One day after seeding, the medium was removed and fresh medium with or without various fatty acids and anti-cancer drug solutions were added, depending on the experimental protocol. The fatty acids were initially dissolved in 95% ethanol and diluted in © Harcourt Brace & Co Ltd 1998

41

such a way that the final concentration of ethanol was not more than 0.02% in all control and fatty acid supplemented cultures. Cell viability was determined by the Trypan blue dye exclusion method. 22 2. Thymidine incorporation studies

To study the growth of various types of tumour cells in the presence of various fatty acids and anti-cancer drugs, the ability of cells to incorporate thymidine as a function of DNA synthesis was used. One day after seeding, 0.5 ~tCi of labelled thymidine (specific activity 18 500 mCi/mmole obtained from BARC, Bombay, India) was added 6 h before harvesting the cells. At the end of the incubation period, the cells were washed at least three times with phosphate-buffered saline (PBS, pH 7.4), detached by trypsinization, extracted for DNA and counted in a liquid scintillation counter. KB-3-1 and KB-ChR-8-5 cells, which are HeLa variant cell lines, were grown and maintained in NUNC culture flasks in bicarbonate buffered DMEM with 10% fetal calf serum and L-glutamine at 37°C in a 5% CO2 humidified incubator as described above. KB-ChR-8-5 cells, which are resistant to the actions of colchicine and vincristine, were grown in the continuous presence of colchicine. The seeding density of the cells, thymidine incorporation studies and Trypan blue dye exclusion method were done as described above. SP 2/0 Ag14, a mouse myeloma cell line, cells were cultured in 80 cm 2 tissue culture flasks (NUNC, Denmark) in RPMI 1640 medium buffered to pH 7.2 and supplemented with 10% human AB serum, 50U/m1 streptomycin and grown at 37°C in a humidified incubator using 95% air and 5% CO2 as described above for KB-3-1 cells. 3. Fatty acid analysis

For fatty acid analysis, 10 x 10 6 KB-3-1 and KB-Chg-8-5 cells were grown in 245 × 245 x 20 mm NUNC tissue culture plates and incubated for 24 h with and without 40 I~g/ml of fatty acids. After incubation, the cells were harvested and analysed by gas-liquid chromatography using Tracor 540 GC as described earlier. 18,22 4. Uptake and efflux of vincristine in HeLa and KB-3-1 and KB-ChR-8-5 cells

The effect of fatty acids on the uptake of radiolabelled vincristine in HeLa ceils was studied using 3H-vincristine sulphate. 1 x 106 cells/ml of medium/weU were seeded in six-well plastic multidishes and grown for 24 h. At the end of 24 h, considered as '0' time, the medium was removed and fresh medium with or without fatty acids (20 ~g/ml)

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 39-54

42

Das et al

was added. Simultaneously the ceils were supplemented with 10 ng/ml of 3H-vincristine (which is equivalent to 1.6nCi/ml) sulphate (specific activity: 6.2Ci mmo1-1 obtained from Amersham International, Amersham, UK). At the end of 12 h of addition, the cells were washed thoroughly with PBS, trypsinized, removed and counted in a liquid scintillation counter to determine the uptake of vincristine by the cells. In the case of KB-3-1 and KB-ChR-8-5 cells, to study the effect of various fatty acids on the uptake of vincristine, the cells were pretreated with fatty acids. To 1 x 104 cells/ ml/well, 1 day after seeding, the medium was replaced with fresh medium along with different doses of fatty acids ranging from 5 to 40 ~g/ml. At the end of 6 h of addition of fatty acids, 50 nM of 3H-vincristine was added and incubated for further time periods. At the end of 1, 2 and 4 h of addition of vincristine, the cells were washed, detached by trypsinization and counted in a liquid scintillation counter. Uptake of vincristine in the presence of fatty acids is expressed as percentage of control. In KB-3-1 and KB-ChR-8-5 cells, the effect of fatty acids on the efflux of vincristine was also studied. 24 h after seeding (1 x 104 cells/mYwell), fresh medium was added along with fatty acids (5-40 ~g/ml) and incubated for 6 h. At the end of the incubation period, 50 nM of 3H-vincristine was added and incubated for another 2 h after which the medium was discarded and cells were washed with PBS. To these cells, 0.5 ml of phenol red free DMEM was added and incubated for an additional 1, 2 and 4 h. At the end of the incubation period, the medium was removed and counted in a liquid scintillation counter. 5. Estimation of anti-oxidants

Since it is now believed that the anti-oxidant capacity of the tumour cells may also determine, to a limited extent, the sensitivity or resistance of cells to the cytotoxic action of various anti-cancer drugs,27,28we studied the effect of various fatty acids on the levels of various anti-oxidants in SP 2/0 cells in vitro.

5b. Estimation of anti-oxidants

The levels of various anti-oxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase and total glutathione in SP 2/0 ceils was determined as described earlier?9-33 6. Estimation of the activity of membrane bound enzymes

The levels of two membrane bound enzymes, 5'-nucleotidase and Na÷-K÷-ATPase, were estimated in fatty acid treated SP 2/0 cells as described earlier. 34 The activity of these enzymes were also estimated in KB-3-1 and KBChR-8-5 cells both before and after treatment with various fatty acids. 7. Estimation of protein kinase C (PKC)

PKC estimation was carried out in polystyrene microtitre (NUNC) plates containing 96 flat bottomed wells. The reaction mixture contained a final concentration of 20mM Tris HC1 pH 7.5, histone type INS 200~g/ml, MgC12 10 mM, CaC12 200 gM, mixed micelles of phosphotidyl serine and diolein (w/v in 0.3% triton X100) in a final volume of 250 ~1. Radiolabelled ATP (specific activity 3000 Ci/mM, 0.1 ~Ci/assay) was added followed by 50 gl of the enzyme sample and incubated for 15 rain at room temperature. The reaction was terminated by adding 100~I of freshly prepared 25% TCA (trichloracetic acid). The samples were cooled on ice for 30 min, filtered over Whatman P-81 phosphoceilulose paper using a NUNC portable cell harvester. Filter paper discs were throughly dried and analysed for radioactivity. Blanks 500O

'~

Concentration

of f a t t y acid

4500 4 0 /zg 4000

3500

3000

5a. Preparation of the cell lysate

2500

SP 2/0 cells seeded in 80 cm 2 culture flasks were allowed to grow to subconfluency at which time fatty acids were added and incubated for 24 h. At the end of the incubation period, the ceils were suspended in Tris-HCl buffer 50 mM, pH 7.2 containing 2 mM EDTA and 1 mM phenyl-methionyl sulphonyl fluoride (PMSF) and lysed by sonication for 5 s in two cycles. The resulting suspension was centrifuged at 3000rpm (revolutions per minute) for 30m in and the nuclear-free clear lysate was used for the estimation of the activity of various anti-oxidants.

2000

1500

1000

500 0

t

2

3

t

2

3

1

2

3

Time in days O Control v Docosahexaenoic acid G Dihomogamma-linolenia acid Arachidonic acid • Eicosapentaenoie acid • Gamma-linolenic acid • Linoleie acid • A]pha-linolenie acid

Fig. 2 Effectof various fatty acids on the growth of HeLa cells in vitro (thymidine incorporationstudies).

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 39-54

© Harcourt Brace & Co Ltd 1998

Can tumour cell drug resistance be reversed by essential fatty acids and their metabolltes?

were run simultaneously in the absence of protein kinase and the radioactivity recovered on the filter paper P-81 was subtracted. The PKC levels were estimated in AK-5 cells with and without fatty acid treatment.

that the percentage of live cells is less as the dose of fatty acids is increased. Of all the fatty acids tested, EPA and DHA were found to be the most potent. Results of the thymidine incorporation data presented in Figure 2 further support the observation that n-6 and n-3 fatty acids have cytotoxic/cytostatic action on HeLa cells. The results shown in Figure 3 indicate that of all the fatty acids tested, only ALA and EPA are the most potent in inhibiting the growth of SP 2/0 cells at all the concentrations tested. DHA and OA were also found to affect the proliferation of SP 2/0 cells but only to a limited extent

RESULTS Effect of fatty acids on the growth and survival of tumour cells in vitro

The effect of various fatty acids on the survival of HeLa cells is given in Figure 1. It is clear from these results 25000

5 /zg/m[

43

20/ig/mI

I O/zg/ml

40/~g/m]

2oooo

15000

.~ 10o00

i

5000

24

48

72

24

48

72

24

48

72

24

48

72

Fig. 3 Effect of various fatty acids on the proliferation of SP 2/0 cells in vitro (thymidine incorporation studies). 50

~

ml ""

25

lO/zg/ml

"

Fatty

acid

40/zg/ml co

10

5

0 [

0 • • •

-

0 • • •

II

II!

[

II Ill I T i m e in d a y s

Control • - • Linoleic acid Di-homo gamma linolenic acid Alpha-linolenic acid Docosa hexaenoic acid

v [] z~ o

II

-

v o ~ o

Ill

I

[I

If[

Gamma-linolenic acid Arachidonic acid Eicosa penf.aenoic acid Oleic acid

Fig. 4 Effect of various fatty acids on the viability of SP 2/0 cells in vitro (cell number).

© Harcourt Brace & Co Ltd 1998

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 39-54

44

Das et al

(Fig. 3). The n-6 fatty acids: LA, GLA, DGLA and AA did not show any inhibitory effect on the proliferation of SP 2/0 cells at all the doses tested. In fact, both DGLA and AA stimulated the proliferation of SP 2/0 cells (Fig. 3). These results are supported by the observation that both ALA and EPA also decreased the viability of SP 2/0 cells in vitro (Fig. 4). DHA, LA, GLA and DGLA have also decreased the viability of SP 2/0 cells but only to a limited extent and that too only at 40 I~g/ml concentration. The effect of vincristine on the survival of KJ]-3-1 and KB-ChR-8-5 cells is given in Figure 5. It is evident from these results that KB-ChR-8-5 ceils are resistant to the cytotoxic action of vincristine compared to the response of KB-3-1 cells. The results shown in Figure 6 suggest that of all the fatty acids tested, EPA and DHA are the most potent in reducing the survival of KB-3-1 and KB-ChR-8-5 cells in vitro. The cytostatic/cytotoxic action of various fatty acids on KB-3-1 and KB-ChR-8-5 cells is further evident from the thymidine incorporation studies, the data of which are given in Figure 7.

120 KB-3-1

100

0 • v

-

0 • v

Day I D a y II D a y Ill

T h e effect of vincristine on the s u r v i v a l o f KB-3-1 and KB-

KB-8-5 •

= 6o o

-

•

ChR-8-5 cells is given in Figure 5. It is clear from these results that KB-ChR-8-5 cells are indeed resistant to the cytotoxic action of vincristine. In Figure 6, the effect of various fatty acids on the survival of KB-3-1 and KB-ChR-8-5 ceils is given. Of all the fatty acids tested, EPA and DHA were found to be the most potent on both the cell lines. The cytotoxic action of fatty acids on KB-3-1 ceils in order of potency was as follows: D H A > EPA > G L A = D G L A > A A > L A > ALA,

Day I

[] -

[] D a y II

•

•

-

Day

III

40

20

0

J 2.5

i 5

Concentration

Ii0

of

J 15

vincristine

HeLa ceils

The effect of vincristine, doxorubicin and cis-platinum on the growth of HeLa ceils in vitro is given in Figure 8. It is evident from these results that all the three anti-cancer drugs are capable of suppressing the growth of HeLa ceils at the concentrations tested. Since both fatty acids and anti-cancer drugs can induce a cytotoxic/cytostatic action on HeLa ceils in vitro, possible synergism in their action was evaluated by the addition of sub-optimal doses of both fatty acids (only GLA and EPA were used as representative of n-6 and n-3 fatty acids) and anti-cancer drugs together to the cells in vitro. The results given in Figure 9 indicate that GLA at 20 ~g/rnl and EPA at 10 ~tg/ml when used alone have little or no action on the growth of HeLa cells at the end of 24 h of incubation. But in the presence of 1-1000 ng/ml of vincristine, doxorubicin and cis-platinum both GLA and EPA augmented the cytotoxic action of the anti-cancer drugs (Fig. 9). It was observed that EPA is more potent than GLA in inducing growth suppression of HeLa ceils in the presence of the anti-cancer drugs. This suggests that the tumoricidal action of anti-cancer drugs can be potentiated by GLA and EPA (EPA > GLA). KB-3-1 and KB-Ch~-8-5 cells

8O

o

Synergism in the action of fatty acids and anti-cancer drugs on tumour cells in vitro

i

20

(nM)

whereas the action o n KB-ChR-8-5 cells w a s as follows:

Fig. 5 Effect of different doses of vincristine on the survival of KB-3-1 and KB-Ch"-8-5 cells in vitro.

DHA

> AA > DCLA

= E P A = G L A > L A > A L A as seen o n

1001 O-

oLA

80

80

60

60 40 20

0 0

Cony. of Fatty acid (/~g/ml)

eGLA

~z -

~

D~LA

•

V

AA

-

[] -

rl ALA

•

•

-

z~ -

20

'°f

O-

1'0 Conc.

:~0

3~

EPA

z~ D H A

410

of Fatty acid (#g/col)

Fig, 6 Effect of various fatty acids on the survival of KB-3-1 and KB-ChR-8-5 cells in vitro on day 3. Prostaglandins, LeukotMenes and Essential Fatty Acids (1998) 58(1), 39-54

© Harcourt Brace & Co Ltd 1998

Can tumour cell drug resistance be reversed by essential fatty acids and their metabolltes? 45

140

140

8

~ T

100!

E~O0~ ~

KB-3-1

AA 120

DGLA

120

i

B0

~

~ 6O N 40

-

0

Dayl

•

-

•

Day2

-

v Day3

60

N

Cells

0

±

40 20

20

Cells

KB-8-5

0

;

0

lo

~o

,0'

15 t0 20 ~0 Cone, of Fatty Acid (]~g/mI)

o

Conc. of Fatty Acid (~g/ml)

•

-

•

Dayl

t 4O

140

t~ -

o Day2

120

120

•

• Day3

1 O0

1001 80

~ ao u

-

60

ioo

40

N 4O N

20

20 .

.

5 Conc. of

.

o

.

20

10

40

Fatty Acid ( ~ g / m l )

,

0

5 Cone

, 10

;

~

2

0

of Fatty Acid ( ~ g / m l )

Fig. 7 Effect of various fatty acids on the ability of KB-3-1 and KB-ChR-8-5 cells to incorporate thymidine in vitro.

2000

2000

Control

1800

~-~

CLA

1600

EPA

1600 1400

1200

1200 800

1000 800

400

600 400

c o n t r o l 10ng/ml ltlOng

lml

cis-PL~TINUM

200

1000 lOng/ral 100 10nK/ml 10Ong 1000 ng/ml ml n /ml VlNCRINI~'E DOXRUflICIN~

*p ~ 0.05 a l compared to control

0

CONTROL

cis-PLAT]NUM

VIN ORIS'TINE

cis p l a t i n u m

vincristine

ing/ml

0 Ing/ml

10ng/ rnl

lng/ml

lOOng/ml lO00ng/ml

Fig. 9 Combined effect of fatty acids (GLA/EPA) and anti-cancer drugs on the growth of HeLa cells in vitro.

DOXORUBICIN

doxorubicin i ng/ml 10ng/ml

lOng/ml

I OOng/ml

100ng/ml

]O00ng/ml

"p • 005 c o m p a r e d to control

Fig. 8 Effect of anti-cancer drugs: vincristine, doxorubicin and cis-platinum on the growth of HeLa cells in vitro. *P < 0.05 compared to control.

day 3 of incubation. These results indicate that both vincristine-sensitive and -resistant cells are almost equal in their sensitivity to the cytotoxic action of various fatty acids. Since vincristine-resistant KB-ChR-8-5 cells are sensitive to the tumoricidal action of fatty acids (as sensitive as that of the vincristine-sensitive KB-3-1 cells), the possible abil© Harcourt Brace & Co Ltd 1998

ity of fatty acids to enhance their sensitivity to the cytotoxic action of vincristine was studied. Results of this study, given in Figure 10, revealed that pre-incubation of KB-ChR-8-5 cells with sub-optimal doses of various fatty acids can render them sensitive to the tumoricidal action vincristine. This is especially true of GLA, AA, EPA and DHA. This suggests that c-UTAs can enhance the sensitivity of drug-resistant KB-ChR-8-5 cells in vitro to the cytotoxic action of vincristine. Thus, the results shown in Figures 9 and 10 indicate that cis-unsaturated fatty acids can sensitize the turnout cells to the cytotoxic actions of various anti-cancer drugs.

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(I), 39-54

46

Das et a~

AA

120

12o

100

[~

o

vincr[.una (SnM)

°o

Vlncr[It[na (lOnN)

400 100

Fatty acid (5/ag/ml)

w

¢¢I

ratL,v .aid lO~s/ml)

E ~ 6O

r--'l vin=rl.tina(SnM) * Fatty aeid (S/q~ml) N~) Vlner~tlna (SnU) + Fatty acid (iO/ag/ml)

~ 40

Vlncrlstirle (lOnR) + [aLLy sold (5/~/ml) • ~

20

DHA

8O

° o m

60

~

40

=~

~nerlsLlne (IOnM) + Fatty ae|d ( l O ~ m l )

~

20

Day I

Day I

Day II Day ][] • AS compared wlth control, p • O.OS

A

Day I|

Day ILl

* As c o m p a r e d with c o n t r o l , p ~ 0 . 0 5

B

"6 120

o~ o 9

O1,A

.J

EPA

120

o

9

1 O0

80

100

] o

8O

oo m

60

6O

40

40

20

20

0 Day l

C

Day II

Day Ill

* As c o m p a r e d with c o n t r o l , p ~ 0 . 0 5

D

Day [[

Day !

Day ][I

* AS c o m p a r e d with control, p ~; 0 . 0 5

Fig. 10 ( A - D ) Effect of sub-optimal doses of vincristine and fatty acids on the proliferation of KB-ChR-8-5 cells in vitro.

Influx and efflux of vincristine in HeLa, KB-3-1 and KB-ChR-8-5 cells in the presence of fatty acids

Table I Effect of cis-UFAs on the uptake of (all) vincristine HeLa cells in vitro

One of the mechanism(s) involved in the development of multi-drug resistance involves decreased intracellular accumulation of the drug due to reduced uptake and/or increased efflux. Studies performed both by us and others 18,2°,22,23,34have suggested that exogenously added fatty acids are incorporated into the cell membranes rather rapidly. Incorporation of c-UFAs into the cell membrane lipids can enhance the membrane fluidity, which can lead to changes in the influx and efflux of drugs by the tumour cells. In order to verify this possibility, the effect of various c-UFAs on the uptake and efflux of radiolabelled vincristine in HeLa, KB-30-1 and KB-Ch~-8-5 cells was studied. Results of this study with HeLa cells are given Table 1. It is evident from these results that GLA, ALA, EPA and DHA can enhance the uptake and consequently the intracellular concentration of vincristine in HeLa cells. This increase in the uptake of vincristine in

Group Vincristine Vincristine Vincristine Vincristine Vincristine Vincristine Vincristine Vincristine

CPM/1 x 10 s cells control + 20 I~g m1-1 GLA + 20 I~g ml-t DGLA + 20 I~g m1-1 LA + 20 pg m1-1 AA + 20 I~g m1-1 ALA + 20 t~g m1-1 EPA + 20 p,g m1-1 DHA

*As compared to control,

752 1617 984 875 989 1124 1256 1825

4- 136 4- 2 1 0 "

± ± ± ± ± ±

103 116 136 108" 129" 243*

P < 0.05.

the presence of c-UFAs may explain the enhanced cytotoxicity of anti-cancer drugs when given along with the fatty acids. The results given in Figure 11 indicate that c-UFAs can enhance the uptake of vincristine and inhibit the drug efflux and thus, fatty acids can augment the intracellular concentration of the anti-cancer drug vincristine, in KB-

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 39-54

© Harcourt Brace & Co Ltd 1998

Can tumour cell drug resistance be reversed by essential fatty acids and their metabolltes? 47

225 =" 2 0 0

T

-

150

e-

@LA

V-

V GLA 125

175

-

•

•

O-

OLA

O-

OOLA

"6

DGLA

V

o

~125

;~-

75

"

:B

-

•

-

i ALA

A

-

A

EPA

•

-

•

DHA

o

• - •

AA

£3

-

[3

ALA

•

-

•

EPA

A

DHA

50 A -

50 I 25 0

0

N I

I

1

Z

A

I 3

DCLA

v

1001

[3 AA

[3-

I

25

0

4

Time in hours

0

B

t2

'l

'3

14

Time in hours

Fig. 11(A) Uptake of vincristine (radiolabelled) in the presence of various cis-unsaturated fatty acids in KB-ChR-8-5 cells in vitro.

Fig. 11(B) Efflux of vincristine in the presence of various cis-unsaturated fatty acids in KB-Chn-8-5 cells in vitro.

ChR-8-5 ceils (similar data were obtained with KB-3-1 hence, are not shown) in vitro. Thus, the ability of c-UFAs to enhance the intraceilular concentration of anti-cancer drugs can ultimately lead to increase in the tumoricidal action of these drugs.

CE (cholesterol ester) fraction. In general, it can be said that KB-ChR-8-5 ceils are deficient in those fatty acids (such as DHA, EPA and GLA) which when supplemented in culture were found to be highly toxic to these cells. Additional studies revealed that the concentrations of GLA, AA, EPA and DHA were increased by anywhere between 2- and 15-fold in the PL, I=FA(free fatty acid) and EL cellular lipid pools of GLA-, AA- and EPA-treated cells respectively in comparison to the untreated cells Gables 1-5) suggesting significant incorporation of the fatty acids added.

Fatty acid analysis of KB-3-1 and KB-ChR-8-5 cells

It is evident from the resuks presented above that fatty acids can reverse tumour cell drug-resistance. In view of this, it is worthwhile to study for any possible differences in the fatty acid composition between drug-sensitive and resistant tumour ceils. Hence, fatty acid analysis of KB-3-1 and KB-ChR-8-5 cells was studied. Results given in Tables 2-6 suggest that drug-resistant KB-Ch~-8-5 cells contain low amounts of GLA, ALA, 22:5 n-3 and DHA in the PL (phospholipid) fraction; 18:0 in the EL (ether lipid) fraction; and higher amounts of DGLA, AA and DHA in the EL fraction and 22:4n-6 and DHA in the

Anti-oxidants in tumour cells

Several studies have demonstrated that the sensitivity or resistance of tumour ceils to the cytotoxic action of anticancer drugs and cytokines is determined, at least in part, by the intracellular anti-oxidant(s) concentrations. 2° For example, glutathione-depleted cells showed increased

Table 2 The percentage distribution of phospholipid fraction of fatty acids of KB-3-1 and KB-CHR-8-5 cells treated with different fatty acids FA

Control

GLA

16:0 18:0 18:1n-9 18:2n-6 18:3n-6 18:3n-3 20:3n-6 20:4n-6 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3

28.6±1.2 24.4±0.8 18.1±0.7 13.4±0.6 1.2±0.4 0.7±0.3 0.6±0.6 4.7±0.3 1.8±0.3 0.9±0.1 1.3±0.0 1.8±0.4 2.2±0.3

25.5±1.3 23.1±0.8 14.3±0.3 7.9±0.1 13.4±0.2" 0.5±0.0 1.7±0.3 5.9±0.6 1.3±0.2 1.7±0.1 1.8±0.2 0.9±0.3 1.9±0.1 1.05

Uns~s~

0.88

KB-3-1 AA

KB-8-5 AA

EPA

DHA

Control

GLA

24.7±1.2 24.0±0.8 15.6±0.4 9.4±0.3 1.4±0.3 0.9±0.2 1.7±0.4 11.8±0.7" 3.4±0.4 0.8±0.6 1.8±0.3 1.8±0.4 2.6±0.2

25.6±0.6 24.3~0.8 13.4±0.6 7.0±0.4 1.7±0.3 0.9±0.4 0.9±0.3 3.9±0.3 13.6±1.2" 1.4±0.7 2.2±0.3 2.4±0.4 2.8±0.7

28.6±0.8 26.9±1.4 17.3±0.8 15.4±0.2 0.8±0.0 0.7±0.2 0.8±0.1 0.9±0.1 0.3±0.1 1.2±0.1 1.3±0.2 0.7±0.2 4.8±0.3*

34.9±1.2 23.6±1.2 17.4±0.7 13.5±0.6 0.4±0.0* 0.3±0.1" 0.5±0.2 3.7±0.6 2.1±0.1 0.6±0.0 0.8±0.1 0.8±0.1" 1.3±0.2"

27.2±1.1 19.8±0.7 13.7±0.4 14.9±0.4 9.6±0.5* 0.3±0.0 1.9±0.4 5.8±0.3 1.7±0.0 0.4±0.1 1.3±0.0 1.2±0.0 2.2±0.1

1.05

1.00

0.79

0.70

1.12

EPA

30.1±0.7 19.4±0.3 12.6±0.4 13.7±0.6 2.4±0.3 0.8±0.1 0.3±0.1 10.3±1.2" 2.7±0.7 3.2±0.3 1.4±0.2 0.7±0.1 3.4±0.2 1.04

DHA

28,6±0.5 28.8±0.8 20.1±0.6 28.2±0.6 14,6±0.8 18.1±0.6 14.7±0.5 13.4±0.5 0.7±0.1 0.2±0.3 0.1±0.0 0.4±0.1 0.4±0.1 0.6±0.1 4.5±0.2 0.8±0.2 9.8±0.7* 0.1±0.0 0.8±0.3 1.4±0.2 0.6±0.0 1.3±0.0 2.3±0.4 0.7±0.0 2.6±0.3 4.9±0.6* 1.04

0.75

All values are expressed as mean + SD (n = 4); *P < 0.001 compared to control.

© Harcourt Brace & Co Ltd 1998

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 39-54

48

Das et al

Table 3 The percentage distribution of free fatty acid fraction of fatty acids of KB-3-1 and KB-8-5 cells treated with different fatty acids

FA

Control

GLA

16:0 18:0 18:1n-9 18:2n-6 18:3n-6 18:3n-3 20:3n-6 20:4n-6 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3

31.3±1.3 30.2±0.6 16.3±0.7 11.8±0.6 0.2±0.1 0.3±0.0 0.3±0,1 2.8±0.1 1.2±0.0 1.2±0.0 0.9±0.1 1.6±0.4 1.2±0.3

30.1±0.9 28.7±0.7 11.8±0.6 5.4±0.7 9.7±0.6* 0.4±0.2 0.3±0.2 2.3±0.5 0.9±0.0 1.3±0.2 0.6±0.2 1.7±0.3 0.6±0.2 0.54

Unsa~at

0.62

KB-3-1 AA

KB-8-5 AA

EPA

DHA

Control

GLA

28.6±0.7 26.3±0.3 13.4±0.2 7.0±0.8 0.4±0.2 0.0±0.0 0.1±0.0 10.1±2.3" 3.4±0.7 4.4±0.6 0.3±0.3 2.8±0.4 3.4±0.7

29.0±0.1 24.4±0.7 14.6±0.5 8.3±0.7 0.9±0.3 0.6±0.1 0.5±0.1 1.9±0.6 9.1±0.6" 0.8±0.6 0.7±0,3 4.6±0.8 4.3±0,3*

32.8±1.1 27.9±0.6 15.1±0.8 13.1±0.1 1.3±0.4 0.5±0.2 0.3±0.0 1.1±0.3 0.7±0.2 0.9±0.2 1.2±0.3 1.3±0.2 3.4±0.2*

36.8±1.3 28.2±0.9 13.4±0.9 11.6±0.7 0.7±0.1 4.5±0.2 0.3±0.0 1.9±0.4 1.2±0.0 1.3±0.1 1.4±0.1 0.9±0.3 1.6±0.2

30.1±0.9 31.0±0.0 23.4±0.7 24.7±0.6 16.8±0.4 14.7±0.3 13.8±0.4 12.8±0.6 7.8±0.5* 1.2±0.3 0.0±0.0 0.8±0.1 0.3±0.1 0.9±0.1 1 . 3 ± 0 . 4 11.4±0.6" 2.3±0.3 2.4±0.4 0.7±0.1 4.1±0.6 1.9±0.2 4.7±0.2* 0.8±0.2 1.2±0.1 0.5±0.1 1.0±0.0

0.82

0.86

0.64

0.53

0.86

EPA

DHA

31,1±0.6 23,4±0.6 12,4±0.4 10,8±0.4 0.8±0.3 0,3±0.1 0.4±0.1 1.6±0.0 11.7±0.3" 1.8±0.0 0.0±0.0 2.6±0.0 2.8±0.2

33.1±1.2 30.9±1.2 14.3±0.6 12.6±0.5 1.4±0.3 0.3±0.1 0.7±0.1 0.7±0.1 0.6±0.0 0.8±0.0 0.9±0.1 1.2±0.4 2.3±0.5

1.19

0.82

0.55

All values are expressed as mean ± SD (n = 4); *P < 0.001 compared to control.

Table 4 The percentage distribution of triglyceride fraction of KB-3-1 and KB-8-5 cells treated with different fatty acids

KB-3-1 AA

FA

Control

GLA

16:0 18:0 18:1n-9 18:2n-6 18:3n-6 18:3n-3 20:3n-6 20:4n-6 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3

31.7±0.6 34.7±0.6 15.3±0.3 10.7±0.3 0.9±0.3 0.2±0.0 0.2±0.0 0.9±1.1 1.7±0.2 1.3±0.2 0.8±0.0 0.7±0.1 0.9±0.2

35.1±1.9 33.7±0.6 15.3±0.3 6.8±0.7 3.4±0.2* 0.3±0.0 0.5±0.1 0.6±0.0 0.9±0.0 1.4±0.1 1.3±0.0 0.6±0.2 0.3±0.0

29.7±0.7 31.4±0.6 16.4±0.4 8.6±0.7 1.1±0.3 0.0±0.0 0.4±0.0 4.8±0.1" 1.4±0.1 2.3±0.2 3.0±0.0 0.6±0.0 0.5±0.1

0.45

0,63

UnsaVsat

0.50

EPA

DHA

Control

GLA

31.3±0,6 35.7±0,7 14.8±0.0 7.5±0.6 0.7±0.3 0.4±0.1 0.4±0.1 1.3±0.2 3.7±0.8 1.0±0.3 0.7±0.3 1.5±0.2 1.7±0.1

33.2±1.6 32.3±0.4 13.4±1.2 12.8±0.2 0.6±0.1 0.6±0.2 0.7±0.1 0.6±0.1 0.4±0.0 0.8±0.2 1.3±0.0 2.2±0.2* 2.1±0.2"

35.6±1,2 31.8±0,9 14.9±0,8 10.7±0.4 0.6±0,2 0.3±0.2 0.3±0.0 0.9±0.0 1.2±0.1 0.6±0.2 1.1±0.0 0.9±0.1 0.9±0.1

25.6±0.8 22.8±0.1 17.3±0.4 18.6±0.4 2.9±0.5 0.7±0.1 0.9±0.3 3.1±0.3 3.8±0.1 0.7±0.0 1.4±0.2 0.7±0.0 0.6±0.0

0,49

0.52

0.47

KB-8-5 AA 31.4±0.6 29.0±0.1 15.8±01.2 9.6±0.6 2.7±0.2 0.4±0.0 1.4±0,1 3.4±0.6* 0.9±0.0 0.8±0.1 1.1±0.2 2.6±0.0 0.4±0.1

0.93

0.64

EPA

DHA

31.1±0.5 30.6±0.7 15.7±0.8 8.3±0.4 0.9±0.2 0.6±0.1 0.7±0.1 1.1±0.0 4.6±0.3* 1.3±0.0 1.2±0.0 2.3±0.2 1.9±0.3

29.5±1.3 28.3±1.2 15.5±0.6 18.1±0.5 0.3±0.0 0.7±0.1 0.5±0.1 0.7±0.1 1.2±0.0 0.8±0.1 1.3±0.1 1.2±0.1 1.8±0.3

0.62

0.~

All values are expressed as mean ± SD (n = 4); *P <: 0.001 compared to control.

Table 5 The percentage distribution of ether lipid fraction of KB-3-1 and KB-8-5 cells treated with different fatty acids

FA

Control

GLA

16:0 18:0 18:1n-9 18:2n-6 18:3n-6 18:3n-3 20:3n-6 20:4n-6 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3

32.3±1.0 28.8±0.3 18.8±0.7 17.7±0.3 1.4±0.0 0.4±0.0 0.9±0.1 6.4±0.3 2.8±0.2 1.4±0.3 1.9±0.1 0.9±0.1 2.9±0.1

22.1±1.9 18.7±0.4 9.7±0.4 7.7±0.2 22.8±1.3" 0.3±0.0 3.4±0.2 7.9±0.2 2.3±0.2 0.9±0.3 1.7±0.1 0.7±0.3 1.9±0.1 1.45

UnsaUsat

0.63

KB-3-1 AA

KB-8-5 AA

EPA

DHA

Control

GLA

26.6±0.3 20.3±0.4 13.6±1.2 13.8±0.3 2.8±0.6 0.5±0.1 0.6±0.1 13.8±0.9" 1.8±0.4 1.8±0.3 2.8±0.0 0.4±0.1 1.1±0.2

24.5±0.6 22.4±0.6 15.6±1.0 11.6±0.3 0.9±0.1 0.5±0.0 0.5±0.0 5.4±0.1 11.2±0.4" 2.0±0.6 0.8±0.0 1.4±0.2 3.3±0.1

29.1±0.6 26.3±0.5 12.1±0.3 9.6±0.3 1.7±0.3 0.5±0.0 0.3±0.1 0.3±0.0 0.8±0.0 0.9±0.1 0.7±0.1 0.7±0.1 16.8±0,3"

25.3±1.2 16.6±1.1 16.3±0.7 14.7±0.3 1.2±0.2 0.7±0.1 4.2±0.3 9.9±0.4 3.4±0.4 1.1±0.0 0.8±0.1 0.7±0.3 4.7±0.2

18.3±0.7 15.5±0.5 11.6±0.4 9.7±0.8 27.4±0.4* 0.5±0.1 5.7±0.0 6.5±0.3 0.7±0.1 0.7±0.2 0.9±0.1 0.4±0.0 1.9±0.2

1.13

1.13

0,80

1.39

1.95

20.3±0.6 14.3±0.3 15.4±0.6 9.6±0.0 0.8±0.0 0.4±0.1 3.9±0.6 27.4±0.6* 1.2±0.3 0.9±0.0 3.2±0.3 1.3±0.4 0.9±0.3 1.87

EPA

DHA

22.6±0.8 15.0±0.4 15.8±0.5 11.8±0.7 1.4±0.3 0.7±0.0 5.0±0.1 6.3±0.2 16.4±0.1" 2.3±0.7 1.6±0.3 0.4±0.0 1.8±0.7

29.3±1.9 28.7±0.4 9.7±0.3 10.3±0.2 1.7±0.0 0.6±0.0 0.8±0.1 0.6±0.1 0.7±0.1 1.2±0.3 1.7±0.3 0.8±0.1 13.4±0.2"

1.66

0.71

All values are expressed as mean ± SD (n = 4); *P < 0.001 compared to control.

Prostaglandins, Leukotrienes and Essentia/ Fatty Acids (1998) 58(1), 39-54

© Harcourt Brace & Co Ltd 1998

Can tumour cell drug resistance be reversed by essential fatty acids and their metabolites?

49

Table 6 The percentage distribution of cholesterol ester fraction of KB-3-1 and KB-8-5 cells treated with different fatty acids

KB-3-1 AA

FA

Control

GLA

16:0 18:0 18:1n-9 18:2n-6 18:3n-6 18:3n-3 20:3n-6 20:4n-6 20:5n-3 22:4n-6 22:5n-6 22:5n-3 22:6n-3

33.4±1.9 27.6±0.4 21.8±0.3 10.3±0.2 0.2±0.0 0.0±0.0 0.2±0.0 1.1±0.1 0.9±0.1 0.8±0.1 1.7±0.0 0.3±0.1 0.4±0.1

27.9±1.0 23.6±0.3 17.8±0.1 19.6±0.4 1.8±0.1" 0.3±0.0 0.2±0.2 0.9±0.1 2.6±0.1 2.3±0.2 1.4±0.1 1.3±0.1 0.2±0.0

Unsaffs~

0.70

28.3±1.3 29.0±0.7 19.6±0.4 13.4±0.0 0.7±0.3 0.3±0.0 0.6±0.1 1.7±0.3 1.2±0.0 0.9±0.1 2,3±0.3 2.0±0.2 0.9±0.1

0.93

EPA

DHA

Control

GLA

29.4±0.7 23.4±0.6 20.1±0.4 15.6±0.7 0.7±0.1 0.0±0.0 0.4±0.2 1.8±0.4 1.3±0.3 1.4±0.6 2.7±0.4 1.8±0.0 0.5±0.1

28.3±1.3 27.2±0.3 19.2±0.1 17.5±0.6 0.3±0.0 0.5±0.0 0.8±0.0 0.7±0.0 0.7±0.1 1.0±0.1 0.7±0.0 0.8±0.0 2.3±0.0*

31.4±1.7 27.6±2.3 21.3±0.9 10.4±0.3 0.2±0.0 0.0±0.0 0.4±0.0 1.4±0.1 0.7±0.0 2.5±0.4 0.7±0.1 1.8±0.3 1.4±0.2

27.6±1.3 27.2±1.4 19.4±0.4 11.1±0.2 0.2±0.0 0.6±0.1 6.2±0.3 1.3±0.3 3.3±0,2 0.5±0,1 1.3±0.3 0.5±0,3 0.5±0,2

0.76

0.88

0.80

0.68

0.81

KB-8-5 AA 25.8±0.6 26.7±0.4 25.8±0.3 12.6±0.3 0.4±0.4 0.7±0.1 0.5±0.0 1.8±0.6 1.0±0.1 2.2±0.2 1.1±0.1 1.3±0.2 1.5±0.1 0.93

EPA

DHA

29.6±0.4 23.7±0.1 22.4±0.2 12.3±0.0 0.6±0.3 0.3±0.1 0.9±0.1 1.7±0.2 2.4±0.4 3.6±0.2 1.2±0.0 2.1±0.2 1.6±0.1

28.2±1,3 28.4±0.6 20.3±0,7 14.8±0,6 0.6±0.2 0.8±0,0 0.7±0,3 0.8±0,1 0.3±0.1 1.4±0.2 1.6±0.3 0.4±0.0 1.7±0.1

0.88

0.76

All values are expressed as mean ± SD (n = 4); *P< 0.001 compared to control.

20

1.0

SOD

16

E: 0.8

12

(D .+J O C~ bj] 0.6

O

CATALASE

E tO

a

0.4

0.2

control l

ALA

0.0

EPA

5 /zg/ml Fatty

control

ALA

EPA

acid

[----] 10 # g / m l Fatty acid p < 0.05 as c o m p a r e d to c o n t r o l FI0. 12 Effeotof ALA and EPA on superoxide dismutase and catalase activities in SP 2/0 cells in vitro at the end of 24 h of incubation.

sensitivity to adriamycin, a5 melphalan, cis-platin and doxorubicin. 36'37Thus, one of the factors that can contribute to tumour cell drug resistance is their anti-oxidant content. In view of this, the effect of c-UFAs on the cellular anti-oxidant defences such as SOD (superoxide dismutase), vitamin E, glutathione and catalase was studied. This study was performed in SP 2/0 cells. Of all the fatty acids tested, only AI~ and EPA exhibited potent cytotoxic action of SP 2/0 cells in vitro. 33 Hence, the effect of ALA and EPA only on the levels of various anti-oxidants in SP 2/0 ceils was studied. Results of this study revealed that ALA and EPA (5 and © Harcourt Brace & Co Ltd 1998

10 gg/ml) treatment can decrease the levels of SOD to a significant degree in SP 2/0 cells (Fig. 12). ALA treatment decreased whereas EPA enhanced the activity of catalase at the end of 24 h of incubation (Fig. 12). A decrease in the levels of glutathione peroxidase (GPX) and glutathione reductase (GR) with ALA (5 and 10 gg/ml) treatment whereas an increase with EPA treatment (10 gg/ml) was observed (Fig. 13). A decrease in GR levels was noted when the cells were treated with EPA (5 gg/ml). A significant change in the levels of glutathione-S-transferase (GST) was induced by ALA (at 5 ~g/ml dose) (Fig. 13). Total glutathione levels were elevated in the cells treated

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 39-54

50

Das et al

OST

OPx

GSH

GR

ii,oo

6

75

300

*

200

~

"°IIIIIII ~

Ocontrol ALA EPA

*

50

0

control ALA EPA

~15

~ 10

i

control ALA--EPA

control A[~. EPA

GST -- Glutathione-S-Transferase ; GPx = Clutathione peroxidase GR = Glutathione Reductase Total GSH = Glutathione I 5/zg/ml Fatty acid p < 0.05 a s c o m p a r e d to c o n t r o l

[-~

lO/zg/rnl F a t t y a c i d

Fig. 13 Effect of ALA and EPA on the anti-oxidant defence molecules of the giutathione cycle in SP 2/0 cells in vitro at the end of 24 h of incubation.

with 10 ~g/ml of ALA and EPA at the end of 24 h of incubation. These resuks indicate that a marked degree of alteration in the levels of various anti-oxidants in SP 2/0 cells can be induced by ALA and EPA treatment. Activity of cell membrane bound enzymes in the tumour cells

The properties of cell membrane will determine the uptake and efflux of anti-cancer drugs by tumour cells. Transport is also influenced by changes in membrane fatty acid composition. Fatty acid modifications are associated with changes in the physical and functional properties of the cell membrane. Hence, the effect of various fatty acids on the activities of cell membrane bound enzymes such as NA+-K+-ATPase and 5'-nucleotidase were studied. In SP 2/0 cells both ALA and EPA inhibited the activities of both the enzymes to a significant degree (Fig. 14). These results suggest a potential role for ALA and EPA in the modification of the functions of membrane bound enzymes of SP 2/0 cells. On the other hand, results given in Table 7 indicate that GLA and EPA can increase the activity of Na÷-K÷ATPase both in KB-3-1 and KB-ChR-8-5 cells. DHA enhanced the activity of the enzyme Na+-K+-ATPase in KB-3-1 cells but not in the KB-Ch~-8-5 cells. These results indicate that c-UFAs can alter the activi-

ties of cell membrane bound enzymes which in t u m may be responsible for changes in the sensitivity or resistance of the tumour cells to the cytotoxic action of various anticancer drugs. This in turn may increase the uptake and/or decrease the efflux of these drugs due to changes in the cell membrane properties induced by c-UFAs. As it is, the activity of both Na÷-K÷-ATPase and 5'-nucleotidase are low in the vincristine-resistant KB-Chg-8-5 cells and were increased to a significant degree by the action of c-UFAs, especially AA and DHA (Table 7). In the presence of these fatty acids the uptake of vincristine was enhanced whereas the efflux was decreased resulting in an increase in the intracellular concentration of the anti-cancer drug. Since fatty acids altered the activities of the cell membrane bound enzymes and of the drug uptake and efflux, it is likely that both these changes are due to alterations in the cell membrane lipid composition. c-UFAs and protein kinase C in tumour cells

is known that rat brain protein kinase C (PKC) can be activated by oxidation products of c-UFAs. 38 McPhail et alw showed that c-UFAs themselves can activate Ca 2+dependent PKC and that this effect is not dependent o n their conversion to cyclooxygenase and lipoxygenase products. These reports coupled with the observation that vitamin E can inhibit PKC activity4° indicates that fatty acids and their peroxidation products can modulate It

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 39-54

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Can tumour cell drug resistance be reversed by essential fatty acids and their metabolltes?

51

900 control

E

800

[]T]]~ALA(5~g/ml)

700

~

ALA (10~g/ml)

~

ALA (20~g/ml)

600

EPA (5~g/ml)

c, 500

EPA (10~g/ml)

400

EPA (80~g/ml)

~o

300

:¢

200

100 0

A

p

24 hours

18 hours 0.05 as compared

to control

Fig. 14(A) Effect of ALA and EPA on the activity of cell membrane bound enzymes in SP 2/0 cells in vitro: NA*-K*-ATPase.

400 m

m~

control

[]]T[~ALA (5/~g/ml)

0

ALA (10/zg/rnl)

300

ALA (20/zg/rnl) m

EPA (5/zg/ml) EPA (10/zg/ml)

200

=-• 100

24 hours

18 hours B

P ~ 0.05 as c o m p a r e d

~-~ EPA (20/~g/ml)

I

to c o n t r o l

Fig. 14(B) Effect of ALA and EPA on the activity of cell membrane bound enzymes in SP 2/0 cells in vitro: 5'-nucleotidase.

PKC activity and thus, may have a regulatory role in tumour cell growth. Hence, the effect of fatty acids on PKC activity in AK-5 tumour cells was studied. The results of this study given in Table 8 suggest that © Harcourt Brace & Co Ltd 1998

PKC activity remained high compared to basal levels both in the cytosolic and particulate fractions upon stimulation and that various fatty acids can enhance the activity of PKC in the AK-5 tumour cells.

Prostaglandins, Leukotrienes and Essential Fatty Acids (1998) 58(1), 39-54

52

Das et a~

Table 7 Effect of c-UFAs on Na+-K*-ATPase and 5'nucleotidase in KB-3-1 and KB-8-5 cells

Control GLA AA EPA DHA

KB-3-1 Na*-K*-ATPase 5'Nucleotldase

KB-8-5 Na*-K÷-ATPase 5'Nucleotldase

0.662 + 0.093 1.012±0.113" 0.932 ± 0.121 * 0.763±0.156" 0.871 ±0.093*

0.120 ± 0.011 0.159±0.031 0.161 ± 0.012" 0.140±0.027 0.127±0.011

0.503 + 0.034 0.712±0.019" 0.860 ± 0.056* 0.602±0.112 0.231 ± 0.031"

0.131 ± 0.040 0.119±0.031 0.191 ± 0.027* 0.163±0.040 0.070±0.009

The enzyme activity was expressed in ~moles of Pi/h/mg protein; *P < 0.05 compared to control,

DISCUSSION

Drug-resistance is a major barrier to effective cancer chemotherapy. Even when a variety of combinations of chemotherapeutics are used, patients eventually exhibit simultaneous resistance to some or all of the drugs, leading ultimately to therapy failure. Drug-resistance arises through a variety of mechanisms. The amazing capacity of the turnout cells to survive and adapt to an unfavourable environment is one of the inherent features of malignancy. Once MDR has developed, be it from initially resistant ceils or from selective pressures by the drug regime, methods need to be designed to overcome this undesirable situation. One of the approaches that may help in reversing drug resistance include identification of drugs/substances that can increase the influx and decrease the efflux of anti-cancer drugs in the turnout cells. The results presented here suggest that essential fatty acids and their metabolites in particular, GLA, DGLA, AA, I~PA and DHA cannot only kill tumour cells in vitro (Figs 1-4 and 6) but are also capable of increasing the anti-cancer drug influx and decreasing the efflux in the tumour ceils (KB-3-1, KB-Chg-8-5 and HeLa cells, Table 1 and Fig. 11). In addition, the results presented here suggest that c-UVAs have the ability to sensitize vincristineresistant ceils to the cytotoxic action of vincristine by augmenting the drug uptake. In a similar fashion, even in HeLa cells GLA and I~PApotentiated the cytotoxic action of anti-cancer drugs (Figs 9 and 10). Since c-UFAs can be incorporated into the cell membrane lipids, it is likely that these fatty acids are able to alter the membrane fluidity and thus, are able to enhance drug-uptake and decrease drug-efflux. This idea is supported by the observation that c-UFAs can alter the activities of cell membrane bound enzymes in SP 2/0 ceils in vitro (Fig. 14). One mechanism by which tumour cells show resistance to the cytotoxic action of anti-cancer drugs includes increased expression of anti-oxidant enzymes, z~ Results presented here (Figs 12 and 13) showed that ALA and EPA can decrease the levels of SOD, catalase and glutathione at least in SP 2/0 cells. This suggests that this could be yet

another mechanism by which c-UFAs are able to reverse tumour cell drug-resistance in addition to their capacity to alter drug influx and efflux. In an earlier study, 21 it was suggested that there is a dose relationship between c-UFAs, anti-cancer drugs and their action on PKC with regard to the sensitivity of tumour ceils to drugs. If this is true, it is anticipated that c-UFAs can modulate the activity of PKC in tumour cells. As predicted, the results shown in Table 8 indicate that various fatty acids can enhance PKC activity in AK-5 cells in vitro. In this context it is interesting to note that there is a dose link between PKC activity and lipid peroxidation process. Gambetta et a141 studied the effect of doxorubicin and its analogues on PKC activation and showed that PKC activation by anthracyclines is mediated by lipid peroxidation since it does not occur in the absence of malondialdehyde formation. This is supported further by the observation that PKC membrane translocation and activation may be important for mediating membrane damage and lipid peroxidation after hepatocytes are exposed to oxygen based radicals. 42 Thus, the ability of c-l_lF/ks to augment lipid peroxidation TM and induce cytotoxicity may, at least, in part be due to their action on PKC activity. Recent studies suggest that p53, a tumour suppressor gene, is an essential component of the apoptic process induced by anti-cancer drugs? Lowe et al5 demonstrated that tumours expressing the p53 gene contained a high proportion of apoptic cells and regressed after treatment with gamma-radiation or anti-cancer drug, adriamycin. On the other hand, p53-deficient tumours showed resistance to treatment suggesting that p53 status may be an important determinant of tumour response to therapy. If this is true, is it possible that c-UFAs can modulate the expression of p53 in turnout or normal ceils? In a recent study, we noted that c-UFAs can induce apoptosis in tumour cells (unpublished data). Tiilotson et a143 showed that rat mammary turnout (NMU) ceils when exposed to DHA show an increase in the expression of p53 which is associated with suppression of cell proliferation. On the other hand, LA which enhanced the growth of NMU cells decreased p53 levels in these cells. These results suggest

Prostaglandins, Leukott~enes and Essential Fatty Acids (1998) 58(1), 39-54

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Can tumour cell drug resistance be reversed by essential fatty acids and their metabolites?

53

Table 8 Effect of fatty acids on PKC activity Treatment (pg/ml)

Unstimulated Cytosol Particulate

Stimulated Cytosol Particulate

Control

0.46 ± 0.83

0.651 ± 0.083

0.717 ± 0.087*

1.5 ± 0.35*

ALA 25 50

0.766 + 0.056 0.821 ± 0 . 1 2

0.808 ± 0.067 1.114±0.20

0.720 ± 0.043 0.848±0.059

1.64 + 0.091" 2.21 ± 0 . 1 6 1 "

GLA 25 50

0.766 ± 0.024 0.765 ± 0.097

1.2 ± 0.19 1.32 ± 0.168

1.18 + 0.16" 1.002 ± 0.042*

2.603 ± 0.242* 2.8 ± 0.477*

AA

25 50

0.715 ± 0.029 0.733 ± 0.066

1.2 ± 0.154 1.12 ± 0.093

1.2 ± 0.067* 1.2 ± 0.053*

2.4 ± 0.03* 2.9 ± 0.252*

EPA 25 50

0.78 ± 0.087 0.77 ± 0.40

1.085 ± 0.062 1.714 ± 0.101

1.3 ± 0.035* 1.343 ± 0.096*

2.4 ± 0.196* 3.5 ± 0.01 *

D HA25 5O

0.780 ± 0.021 0.763 ± 0.1

1.1 ± 0.048 1.53 ± 0.164

1.04 ± 0.09* 1.5 ± 0.064"

2.4 ± 0.205* 2.53 ± 0.162"

P < 0.05 compared to control

that one m e c h a n i s m b y w h i c h c-UFAs are able to suppress the g r o w t h of t u m o u r cells, induce apoptosis and e n h a n c e their sensitivity to the cytotoxic action of various anti-cancer drugs m a y include their ability to e n h a n c e the expression of p53. In summary, the results of our results presented here and elsewhere indicate that c-UFAs are not only cytotoxic b u t that are also capable of reversing t u m o u r cell drug resistance and e n h a n c e the sensitivity of t u m o u r cells to the tumoricidal action of various anti-cancer drugs. Some of the m e c h a n i s m s b y w h i c h these fatty acids are able to reverse drug resistance or e n h a n c e their sensitivity to anti-cancer drugs include: 1. 2. 3. 4. 5. 6.

e n h a n c e d uptake and decreased drug efflux, alterations in cell m e m b r a n e fluidity, suppression in the activity of anti-oxidant defences, modification of the activity of PKC, upregulation of p53 gene expression, a u g m e n t a t i o n of free radical generation and lipid peroxidation process in the t u m o u r cells, ls,22

In addition, drug-resistant t u m o u r cells seem to have low levels of ALA, GLA and DHA (Table 2) in their phospholipid fraction. These results suggest that some, if not all, c-UFAs m a y find use as potential anti-cancer drugs either b y themselves or in combination with the currently available anti-cancer drugs. 44 This is not a distant possibility. In fact, we have s h o w n recently that GLA can be used in the treatment of h u m a n brain gliomas. 4s Hence, more studies are n e e d e d to exploit c-UFAs as potential anti-cancer drugs and to reverse multi-drug resistance in cancer chemotherapy. ACKNOWLEDGEMENTS

Some of the work reported here was supported by grants from the Indian Council of Medical Research, Department of Science © Harcourt Brace & Co Ltd 1998

and Technology, India and Scotia Pharmaceuticals Limited, UK, to Dr U.N. Das. N.M., G.S.K., M.P., and P.S. were all graduate students during the period of this study.

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© Harcourt Brace & Co Ltd 1998