Growth reduction in glioma cells after treatment with tetradecylthioacetic acid: changes in fatty acid metabolism and oxidative status

doi:10.1016/S0006-2952(01)00525-1

Biochemical Pharmacology

Volume 61, Issue 6, 15 March 2001, Pages 639-649

https://doi.org/10.1016/S0006-2952(01)00525-1 Get rights and content

Abstract

During aerobic metabolism, a small amount of partially reduced oxygen is produced, yielding reactive oxygen species (ROS). Peroxisomes and mitochondria are major contributors to cellular ROS production, which is normally balanced by consumption by antioxidants. The fatty acid analogue tetradecylthioacetic acid (TTA) promotes mitochondrial and peroxisomal proliferation, and may induce oxidative stress and change the growth potential of cancer cells. In the present study, we found that TTA reduced [³H]thymidine incorporation in the glioma cell lines BT4Cn (rat), D54Mg (human), and GaMg (human) in a dose- and time-dependent manner. The 50% inhibitory TTA doses were approximately 125 μM for BT4Cn and D54Mg cells and 40 μM for GaMg cells after 4 days. α-Tochopherol counteracted this inhibition in GaMg cells. TTA enhanced the oxidation of [1-¹⁴C]palmitic acid, which could be explained by stimulation of enzymes involved in peroxisomal (fatty acyl-CoA oxidase) and/or mitochondrial (carnitine palmitoyltransferase) fatty acid oxidation. The glutathione content and the activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase were differentially affected. Increased malondialdehyde (MDA) production was seen in TTA-treated GaMg and D54Mg cells, but not in BT4Cn cells, in vitro. In BT4Cn tumor tissue from TTA-treated rats, MDA was increased while the α-tocopherol content tended to decrease. TTA increased the level of cytosolic cytochrome c in BT4Cn cells, which suggests induction of apoptotic cascades. Although several mechanisms are likely to be involved in the TTA-mediated effects on growth, we propose that modulation of cellular redox conditions caused by changes in fatty acid metabolism may be of vital importance.

Introduction

¹Several compounds modulating cellular lipid metabolism also affect the growth of cancer cells. Such compounds include fatty acids such as EFAs [1], [2], [3], [4], [5], [6], [7], [8] as well as more complex compounds such as fibrates and aromatic fatty acids [9], [10]. The cytotoxic potential of the EFAs depends on the number and localization of double bonds and the chain length [11], [12]. A commonly suggested mechanism behind the EFA-mediated antitumoral influence is generation of oxidative stress and production of lipid peroxides. Reduced growth, or loss in cell viability, may result from the production of peroxides or other ROS that affect specific cellular pathways or irreversibly injure the cell.

For several years, we have investigated the metabolic effects of a novel sulfur-substituted fatty acid analogue, tetradecylthioacetic acid (CH₃-(CH₂)₁₃-S-CH₂-COOH), that has major impacts on cellular metabolism [13], [14]. This analogue has many chemical and physical properties in common with normal saturated fatty acids, but it cannot undergo β-oxidation [15]. It is established that TTA is a ligand for nuclear receptors in the PPAR family [16], [17]. The PPARs are reported to contribute to the regulation of differentiation, proliferation, and apoptosis [18]. In rat liver, TTA leads to increased amounts of mitochondria and peroxisomes [19], [20], and the oxidation of fatty acids are enhanced [21], [22], [23].

In rats, TTA has been found to increase the hepatic activity of the peroxisomal H₂O₂-producing fatty acyl-CoA oxidase (FAO), and this was followed by increased lipid peroxidation [24]. The hepatic content of glutathione in TTA-treated rats increased, while the activities of glutathione peroxidase, glutathione S-transferase, and glutathione reductase decreased [24], [25]. Our work was previously largely focused on physiologically normal cells such as hepatocytes, but it was recently found that TTA has antiproliferative properties in human breast cancer cells (MCF-7) [4], [26]. In the present work, we expanded our research on cancer cells and studied the effects of TTA in three glioma cell lines.

It is well documented that mitochondria are major producers of ROS [27], and this may affect their pivotal role in the regulation of apoptosis [28], [29]. The mitochondrial production of ROS is closely related to energy metabolism, associated as it is with respiratory activity. It is likely that TTA affects ROS generation by modulating the mitochondrial and/or peroxisomal oxidative function. TTA itself is reported to possess antioxidant properties in vitro due to the sulfur atom [30]. In this study, we investigated the effects of TTA on cell growth, fatty acid metabolism, and the redox situation in cancer cells.

Section snippets

Chemicals

l-[methyl-¹⁴C] Carnitine hydrochloride and [³H]thymidine (TRA 310) were purchased from Amersham International. [1-¹⁴C]Palmitic acid (50 mCi/mmol) was obtained from New England Nuclear. TTA was prepared at the Department of Chemistry, University of Bergen, as previously described [31]. All other chemicals and solvents were of reagent grade from common commercial sources.

Cells

The rat glioma cell line BT4Cn [32] and the human glioma cell lines D54Mg and GaMg [33] were routinely kept in a standard

Cell growth

The presented results demonstrate that TTA reduced [³H]thymidine incorporation in the two human glioma lines (GaMg and D54Mg) and in the rat glioma line (BT4Cn) (Fig. 1). GaMg cells appeared to be most sensitive, showing 50% inhibition in [³H]thymidine incorporation at 40 μM TTA after 4 days of treatment (Fig. 1A), while a 50% reduction in [³H]thymidine incorporation was found at approximately 125 μM TTA in both D54Mg and BT4Cn (Fig. 1, B and C, respectively). The TTA concentration giving a

Discussion

This study demonstrates that TTA reduces the growth of glioma cells, as can be seen from the reduced [³H]thymidine incorporation and the reduction in cell number (Fig. 1). However, the cell lines differed in their degree of sensitivity. GaMg cells clearly appeared most sensitive, while D54Mg and BT4Cn cells tolerated higher doses (Fig. 1). Different mechanisms seemed to be involved in the TTA-mediated growth reduction in the different cell lines.

All three glioma cell lines were able to oxidize

Acknowledgements

The authors would like to acknowledge the technical contribution of Kari Helland Mortensen, Bjørn Netteland, Svein Kryger, and Liv Kristine Øysæd. We are also grateful to Professor Rolf Bjerkvig for his assistance and for providing the cell lines. This work was supported by the Research Council of Norway and Kaptein L.A. Hermansen og Hustru I. Hermansens legat.

References (54)

M.E Begin et al.
Selective killing of human cancer cells by polyunsaturated fatty acids
Prostaglandins Leukot Med
(1985)
U.N Das et al.
Local application of gamma-linolenic acid in the treatment of human gliomas
Cancer Lett
(1995)
T Pineau et al.
Activation of a human peroxisome proliferator-activated receptor by the antitumor agent phenylacetate and its analogs
Biochem Pharmacol
(1996)
W.G Jiang et al.
Essential fatty acidsmolecular and cellular basis of their anti-cancer action and clinical implications
Crit Rev Oncol Hematol
(1998)
R.K Berge et al.
Impact of cytochrome P450 system on lipoprotein metabolism. Effect of abnormal fatty acids (3-thia fatty acids)
Pharmacol Ther
(1994)
S Skrede et al.
Thia fatty acids, metabolism and metabolic effects
Biochim Biophys Acta
(1997)
E Raspè et al.
Modulation of rat liver apolipoprotein gene expression and serum lipid levels by tetradecylthioacetic acid (TTA) via PPARalpha activation
J Lipid Res
(1999)
J.P Vanden Heuvel
Peroxisome proliferator-activated receptorsa critical link among fatty acids, gene expression and carcinogenesis
J Nutr
(1999)
R.K Berge et al.
Alkylthio acetic acids (3-thia fatty acids)—a new group of non-beta-oxidizable peroxisome-inducing fatty acid analogues—II. Dose–response studies on hepatic peroxisomal- and mitochondrial changes and long- chain fatty acid metabolizing enzymes in rats
Biochem Pharmacol
(1989)
L Froyland et al.
A hypolipidemic peroxisome proliferating fatty acid induces polydispersity of rat liver mitochondria
Biol Cell
(1996)

D Asiedu et al.

Fatty acid metabolism in liver of rats treated with hypolipidemic sulphur-substituted fatty acid analogues

Biochim Biophys Acta

(1990)

S Skrede et al.

The effects of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes

Biochim Biophys Acta

(1989)

S Skrede et al.

Acylcarnitine formation and fatty acid oxidation in hepatocytes from rats treated with tetradecylthioacetic acid (a 3-thia fatty acid)

Biochim Biophys Acta

(1993)

A Demoz et al.

Relationship between peroxisome-proliferating sulfur-substituted fatty acid analogs, hepatic lipid peroxidation and hydrogen peroxide metabolism

Biochem Pharmacol

(1993)

A Demoz et al.

Modulation of plasma and hepatic oxidative status and changes in plasma lipid profile by n-3 (EPA and DHA), n-6 (corn oil) and a 3-thia fatty acid in rats

Biochim Biophys Acta

(1994)

G Kroemer et al.

Mitochondrial control of apoptosis

Immunol Today

(1997)

O Spydevold et al.

Induction of peroxisomal beta-oxidation in 7800 C1 Morris hepatoma cells in steady state by fatty acids and fatty acid analogues

Biochim Biophys Acta

(1989)

W.E Balch et al.

Characterization of protein transport between successive compartments of the Golgi apparatusasymmetric properties of donor and acceptor activities in a cell-free system

Arch Biochem Biophys

(1985)

J Bremer

The effect of fasting on the activity of liver carnitine palmitoyltransferase and its inhibition by malonyl-CoA

Biochim Biophys Acta

(1981)

L Madsen et al.

Docosahexaenoic and eicosapentaenoic acids are differently metabolized in rat liver during mitochondria and peroxisome proliferation

J Lipid Res

(1998)

W.H Habig et al.

Glutathione S-transferases. The first enzymatic step in mercapturic acid formation

J Biol Chem

(1974)

A.M Svardal et al.

Determination of reduced, oxidized, and protein-bound glutathione in human plasma with precolumn derivatization with monobromobimane and liquid chromatography

Anal Biochem

(1990)

S.J Bakker et al.

Cytosolic triglycerides and oxidative stress in central obesitythe missing link between excessive atherosclerosis, endothelial dysfunction, and beta-cell failure?

Atherosclerosis

(2000)

M.B Paumen et al.

Inhibition of carnitine palmitoyltransferase I augments sphingolipid synthesis and palmitate-induced apoptosis

J Biol Chem

(1997)

M.B Paumen et al.

Direct interaction of the mitochondrial membrane protein carnitine palmitoyltransferase I with Bcl-2

Biochem Biophys Res Commun

(1997)

X Liu et al.

Induction of apoptotic program in cell-free extractsrequirement for dATP and cytochrome c

Cell

(1996)

M.E Begin et al.

Differential killing of human carcinoma cells supplemented with n-3 and n-6 polyunsaturated fatty acids

J Natl Cancer Inst

(1986)

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Growth reduction in glioma cells after treatment with tetradecylthioacetic acid: changes in fatty acid metabolism and oxidative status

Abstract

Introduction

Section snippets

Chemicals

Cells

Cell growth

Discussion

Acknowledgements

Prostaglandins Leukot Med

Cancer Lett

Biochem Pharmacol

Crit Rev Oncol Hematol

Pharmacol Ther

Biochim Biophys Acta

J Lipid Res

J Nutr

Biochem Pharmacol

Biol Cell

Biochim Biophys Acta

Biochim Biophys Acta

Biochim Biophys Acta

Biochem Pharmacol

Biochim Biophys Acta

Immunol Today

Biochim Biophys Acta

Arch Biochem Biophys

Biochim Biophys Acta

J Lipid Res

J Biol Chem

Anal Biochem

Atherosclerosis

J Biol Chem

Biochem Biophys Res Commun

Cell

Differential killing of human carcinoma cells supplemented with n-3 and n-6 polyunsaturated fatty acids

J Natl Cancer Inst