Cancer Chemother Pharmacol (1992) 31:79-80
ancer hemotherapyand harmacology 9 Spfinger-Verlag 1992
Short communication
Adriamycin-induced inhibition of arachidonate incorporation into phospholipids of mastocytoma P815 cells Irina V. Kondakova, Svetlana V. Vereschagina, and Evgeny V. Borunov Department of Immunology, Institute of Oncology, Tomsk Scientific Centre, Russian Academy of Medical Sciences, Tomsk 634001, Russia Received 1 February 1992/Accepted 29 April 1992
Summary. A d r i a m y c i n inhibited [1-14C]-arachidonic acid uptake b y m a s t o c y t o m a P815 ceils in a d o s e - d e p e n d e n t m a n n e r (5 - 120 ma). The incorporation o f [1-14C]-arachidonic acid into p h o s p h o l i p i d s was d e c r e a s e d m a i n l y into cardiolipin and p h o s p h a t i d y l c h o l i n e .
Introduction T h e m e c h a n i s m o f the m e m b r a n o t o x i c action o f A d r i a m y cin ( A D R ) , a v e r y effective antineoplastic agent, has not b e e n clarified. R e c e n t studies indicate that A D R alters such m e m b r a n e properties as fluidity [6], p e r m e a b i l i t y [2], and transport o f ions [1] and induces the p e r o x i d a t i o n o f m e m brane lipids [5]. H o w e v e r , the structural and functional properties o f cellular m e m b r a n e s d e p e n d on p h o s p h o l i p i d m e t a b o l i s m , and p h o s p h o l i p i d turnover m a y p o s s i b l y be i n v o l v e d in A D R - i n d u c e d t u m o r - c e l l toxicity. In this report w e p r o v i d e e v i d e n c e that p h o s p h o l i p i d r e a c y l a t i o n can be m o d i f i e d b y A D R .
Materials and methods Cell cultures. Transformed murine mastocytoma P815 mast cells were transplanted i. p. weekly into DBA/2 mice. For in vitro assay, tumor cells were harvested from the abdominal cavity of tumor-bearing mice. Finally, the cells were suspended in 199 medium.
bovine serum albumin, cellular radioactivity was counted in a Mark-3 scintillation counter (Tracor Analytic, USA). Study of AA incorporation (0.2 gCi) into cellular phospholipids was carried out under similar conditions. Phospholipids were extracted according to the method of Folch et al. [4] and separated by high-performance liquid chromatography (HPLC; Ultrachrom G Ti, LKB, Sweden) on a 4.6- x 250-ram TSK-Si150 Ultropac column (particle size, 5 gin). Elation was carried out using 100% solvent A (hexane/2-propanol; 80:20, v/v) for 5 min followed by continuous gradient from 100% solvent A to 100% solvent B (hexane/2propanol/water; 39:52: 9, by vol.) for 30 min; the flow rate was 1 ml/min. Fractions of the eluate were collected every 60 s for the measurement of radioactivity. Aliquots were taken for the determination of phospholipid phosphorus (Pi) by a micromethod [7].
Results and discussion T h e d o s e - d e p e n d e n t effect o f A D R on the A A uptake b y P815 cells is p r e s e n t e d in T a b l e 1. Cells were pretreated for 1 h with 5, 24, or 120 nM A D R prior to their incubation with A A . E x p o s u r e o f m a s t o c y t o m a P815 cells to exogenous [1-14C]-AA resulted in the i n c o r p o r a t i o n o f 14C label into cardiolipin (CL), p h o s p h a t i d y l e t h a n o l a m i n e , phosphatidylinositol, and phosphatidylcholine (PC; Fig. 1). As shown in the c h r o m a t o g r a m s , labeling of the tumor cells in the presence o f A D R (120 riM) led to a reduction o f [1-14C]-AA i n c o r p o r a t i o n m a i n l y into CL.
Table 1. Effect of ADR on the uptake of [1-14C]-AAby mastocytoma P815 cells
Assay of [1-14C]-arachidonic acid uptake by cells and incorporation into phosphotipids. For determination of [ 1-14C]-arachidonic acid (AA) uptake, tumor cells ( 105) were incubated with 199 medium containing ADR (Farmitalia Carlo Erba, Italy) at concentrations of 120, 24, and 5 nu for 1 h at 37 ~C, and 0.05 gCi [1-t4C]-AA (Amersham, UK) was added for a 5-rain period. After two washes with AA-free medium containing 0.1%
Correspondence to: I. V. Kondakova, Department of Immunology, Instirut of Oncology, Tomsk Sci. Centre, Acad. Meal. Sci, 5 Kooperativny Str., Tomsk 634001, Russia
ADR in medium (n~)
Radioactivity (dpm/155 cells)
% Control
0 120 24 5
17,309• 8,452 • 11,486 • 13,610•
100 48.8 66.3 72.8
2,391 1,095" 1,728" 1,191
Cells were incubated with different concentrations of ADR for 1 h and then labelled with 0.05 gCi [1-t4C]-AA for 5 rain. Data represent mean values +_ SD for 8 experiments. * P
ancer hemotherapyand harmacology 9 Spfinger-Verlag 1992
Short communication
Adriamycin-induced inhibition of arachidonate incorporation into phospholipids of mastocytoma P815 cells Irina V. Kondakova, Svetlana V. Vereschagina, and Evgeny V. Borunov Department of Immunology, Institute of Oncology, Tomsk Scientific Centre, Russian Academy of Medical Sciences, Tomsk 634001, Russia Received 1 February 1992/Accepted 29 April 1992
Summary. A d r i a m y c i n inhibited [1-14C]-arachidonic acid uptake b y m a s t o c y t o m a P815 ceils in a d o s e - d e p e n d e n t m a n n e r (5 - 120 ma). The incorporation o f [1-14C]-arachidonic acid into p h o s p h o l i p i d s was d e c r e a s e d m a i n l y into cardiolipin and p h o s p h a t i d y l c h o l i n e .
Introduction T h e m e c h a n i s m o f the m e m b r a n o t o x i c action o f A d r i a m y cin ( A D R ) , a v e r y effective antineoplastic agent, has not b e e n clarified. R e c e n t studies indicate that A D R alters such m e m b r a n e properties as fluidity [6], p e r m e a b i l i t y [2], and transport o f ions [1] and induces the p e r o x i d a t i o n o f m e m brane lipids [5]. H o w e v e r , the structural and functional properties o f cellular m e m b r a n e s d e p e n d on p h o s p h o l i p i d m e t a b o l i s m , and p h o s p h o l i p i d turnover m a y p o s s i b l y be i n v o l v e d in A D R - i n d u c e d t u m o r - c e l l toxicity. In this report w e p r o v i d e e v i d e n c e that p h o s p h o l i p i d r e a c y l a t i o n can be m o d i f i e d b y A D R .
Materials and methods Cell cultures. Transformed murine mastocytoma P815 mast cells were transplanted i. p. weekly into DBA/2 mice. For in vitro assay, tumor cells were harvested from the abdominal cavity of tumor-bearing mice. Finally, the cells were suspended in 199 medium.
bovine serum albumin, cellular radioactivity was counted in a Mark-3 scintillation counter (Tracor Analytic, USA). Study of AA incorporation (0.2 gCi) into cellular phospholipids was carried out under similar conditions. Phospholipids were extracted according to the method of Folch et al. [4] and separated by high-performance liquid chromatography (HPLC; Ultrachrom G Ti, LKB, Sweden) on a 4.6- x 250-ram TSK-Si150 Ultropac column (particle size, 5 gin). Elation was carried out using 100% solvent A (hexane/2-propanol; 80:20, v/v) for 5 min followed by continuous gradient from 100% solvent A to 100% solvent B (hexane/2propanol/water; 39:52: 9, by vol.) for 30 min; the flow rate was 1 ml/min. Fractions of the eluate were collected every 60 s for the measurement of radioactivity. Aliquots were taken for the determination of phospholipid phosphorus (Pi) by a micromethod [7].
Results and discussion T h e d o s e - d e p e n d e n t effect o f A D R on the A A uptake b y P815 cells is p r e s e n t e d in T a b l e 1. Cells were pretreated for 1 h with 5, 24, or 120 nM A D R prior to their incubation with A A . E x p o s u r e o f m a s t o c y t o m a P815 cells to exogenous [1-14C]-AA resulted in the i n c o r p o r a t i o n o f 14C label into cardiolipin (CL), p h o s p h a t i d y l e t h a n o l a m i n e , phosphatidylinositol, and phosphatidylcholine (PC; Fig. 1). As shown in the c h r o m a t o g r a m s , labeling of the tumor cells in the presence o f A D R (120 riM) led to a reduction o f [1-14C]-AA i n c o r p o r a t i o n m a i n l y into CL.
Table 1. Effect of ADR on the uptake of [1-14C]-AAby mastocytoma P815 cells
Assay of [1-14C]-arachidonic acid uptake by cells and incorporation into phosphotipids. For determination of [ 1-14C]-arachidonic acid (AA) uptake, tumor cells ( 105) were incubated with 199 medium containing ADR (Farmitalia Carlo Erba, Italy) at concentrations of 120, 24, and 5 nu for 1 h at 37 ~C, and 0.05 gCi [1-t4C]-AA (Amersham, UK) was added for a 5-rain period. After two washes with AA-free medium containing 0.1%
Correspondence to: I. V. Kondakova, Department of Immunology, Instirut of Oncology, Tomsk Sci. Centre, Acad. Meal. Sci, 5 Kooperativny Str., Tomsk 634001, Russia
ADR in medium (n~)
Radioactivity (dpm/155 cells)
% Control
0 120 24 5
17,309• 8,452 • 11,486 • 13,610•
100 48.8 66.3 72.8
2,391 1,095" 1,728" 1,191
Cells were incubated with different concentrations of ADR for 1 h and then labelled with 0.05 gCi [1-t4C]-AA for 5 rain. Data represent mean values +_ SD for 8 experiments. * P
