Elsevier

Biochemical Pharmacology

Volume 65, Issue 12, 15 June 2003, Pages 1979-1989
Biochemical Pharmacology

Toxic effects of copper-based antineoplastic drugs (Casiopeinas®) on mitochondrial functions

https://doi.org/10.1016/S0006-2952(03)00212-0Get rights and content

Abstract

To elucidate some of the subcellular and biochemical mechanisms of toxicity of metal-based antineoplastic drugs, mitochondria and cells were exposed to Casiopeinas®, a new class of copper-based compounds with high antineoplastic activity. The rates of respiration and swelling, the H+ gradient, and the activities of succinate (SDH) and 2-oxoglutarate dehydrogenases (2-OGDH) and ATPase were measured in mitochondria isolated from rat liver, kidney, heart, and hepatoma AS-30D. Also, oligomycin-sensitive respiration and ATP content in hepatoma AS-30D cells were determined. Casiopeinas® (CS) II-gly and III-i inhibited the rates of state 3 and uncoupled respiration in mitochondria. CS II was 10 times more potent than CS III. The sensitivity to CS II was 4–5-fold higher in mitochondria incubated with 2-OG than with succinate. Thus, at low concentrations (≤10 nmol (mg protein)−1; 10 μM), CS II disturbed mitochondrial functions only when 2-OG was present, due to a specific inhibition of 2-OGDH. At high concentrations (≥15 nmol (mg protein)−1), CS II-induced stimulation of basal respiration, followed by a strong inhibition, which correlated with K+-dependent swelling and cytochrome c release, respectively; K+-channel openers induce a similar mitochondrial response. Mitochondria from liver, kidney and hepatoma showed a similar sensitivity towards CS II, whereas heart mitochondria were more resistant. Oxidative phosphorylation and ATP content were also decreased in tumor cells by CS II. The data suggested that CS affected several different mitochondrial sites, bringing about inhibition of respiration and ATP synthesis, which could compromise energy-dependent processes such as cellular duplication.

Introduction

The success of cisplatin in the treatment of several solid tumors, particularly testicular cancer [1], [2], [3], which was unfortunately accompanied by severe side effects [4], [5], [6], prompted the search and development of new metal-based antineoplastic drugs with diminished toxic effects.

The predominant cisplatin toxic effect in both humans and laboratory animals involves renal tubular dysfunction, degenerative changes, and necrosis (reviewed by Goldstein and Mayor [7]). At the subcellular level, several morphological alterations including mitochondrial swelling, chromatin condensation, and microvilli loss, develop in kidney after cisplatin administration [4], [8]. Kidney tubular epithelial cells contain a large number of mitochondria, and thus some of the cisplatin toxic effects seem to involve a direct interaction with these organelles. Indeed, the cisplatin concentration in renal cortical slices is at least five times higher than that in plasma [9]. Intracellularly, cisplatin is located in all subcellular fractions, but it is mainly concentrated in cytosol, microsomes and mitochondria [9], [10], [11]. This last observation indicates an active uptake of the cisplatin cation by cellular energy-dependent processes, which generate negative-inside transmembrane electric gradients.

Effects of cisplatin on the functions of kidney mitochondria, either isolated or inside intact cells, comprise collapse of membrane potential, disturbance of Ca2+ homeostasis, inhibition of ADP-stimulated (state 3) respiration, and enhancement in the formation of reactive oxygen species [8], [12], [13]. Tumor mitochondria also undergo similar alterations induced by cisplatin [14], [15], [16]. However, it has been proposed that mitochondria might not be a primary target for cisplatin and derivatives [17], since mitochondria are only marginally impaired by micromolar cisplatin concentrations, which are in the range of concentrations used in patients.

Despite this controversy, the observations on alterations of mitochondrial function may have clinical relevance. Mitochondria seem involved in tumorigenesis, through mutagenesis by transfer and insertion of mitochondrial DNA into nuclear DNA, or altered expression and mutation of mitochondrial DNA-encoded proteins. Mitochondria also seem involved in the maintenance of the malignant phenotype by still unknown mechanisms, perhaps related to ATP production [18], [19]. Several different drugs, including polycyclic aromatic compounds [20], [21], aflatoxin [22], cisplatin [23], [24], and perhaps bleomycin [25], preferentially bind to mitochondrial DNA than to nuclear DNA. The naked (histone-lacking) mitochondrial DNA structure facilitates the access of drugs, and its limited repair mechanisms enhance mutation rate and permanent damage induced by drugs and oxidative stress [19], [26]. The development of resistance to cisplatin in many tumor cell lines also seems related to mitochondrial alterations [27].

Seeking less toxic metal-based antineoplastic drugs, it was thought that the design and synthesis of mixed chelate copper-based drugs should bring about such a result, considering that copper is an essential metal ion [28]. Some of these copper-based drugs called Casiopeinas® have exhibited greater antineoplastic potency than cisplatin in in vitro and in vivo studies of a variety of tumor cell lines [28], [29]. In addition, Casiopeinas® also show superoxide dismutase-like activity [30] and a low potency to induce genomic instability through intrachromosomal recombination [31]; these features suggest that these drugs have diminished undesirable side effects. However, the toxicological effects of Casiopeinas® on mitochondrial functions have not been evaluated as yet.

Section snippets

Chemicals

The following compounds were acquired from Sigma: sucrose, EGTA, protease Nagarse, digitonin 50%, safranin O, dithiothreitol (DTT), thiamine pyrophosphate, 2,6-dichlorophenol indophenol (DCPIP), phenazine methosulfate (PMS), Triton X-100, CCCP, pyruvate, 2-OG, succinate, glutamate, malate, glucose-6-phosphate dehydrogenase, rotenone, antimycin, and oligomycin. NADH, NAD+, NADP+, ADP, ATP, coenzyme A, hexokinase, and valinomycin were from Boehringer. Mops and Hepes were from Research Organics.

Respiration and membrane potential

ADP-stimulated (state 3) respiration was inhibited by both CS II or III in a time-dependent fashion, in mitochondria incubated with 2-OG (Fig. 2A) or succinate (not shown) as substrate. Similarly, membrane potential collapsed in the presence of CS in a time- and concentration-dependent manner (Fig. 2B). To establish the sensitivity of liver and hepatoma mitochondria towards CS, respiration was measured after 4 min of preincubation with CS II using two different oxidizable substrates. The rates

Discussion

The data of the present work show that CS may directly interact with mitochondria, isolated or within intact cells, inducing a variety of effects on different sites, which brings about inhibition of oxidative phosphorylation and, eventually, cellular ATP depletion. The most pronounced CS II effect, at concentrations below 10 nmol (mg protein)−1, was the strong inhibition of 2-OGDH through a reaction with the CoA thiol group. As the pyruvate dehydrogenase complex also requires free CoA, it is

Acknowledgements

This work was partially supported by Grant G 35012-N from CONACyT-México. The authors thank Dr. G. MacCarthy for English corrections.

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