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FLÁVIA ALESSANDRA GUARNIER
ESTRESSE OXIDATIVO EM MÚSCULO ESQUELÉTICO DE RATOS
COM CAQUEXIA INDUZIDA POR TUMOR DE WALKER-256.
LONDRINA
Dissertação apresentada ao Curso de Pós-
graduação em Patologia Experimental,
Departamento de Ciências Patológicas da
Universidade Estadual de Londrina, como
requisito final à obtenção do título de mestre.
Orientador: Dr. Rubens Cecchini.
2006
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Este é um trabalho realizado no laboratório de Fisiopatologia de Radicais Livres, da
Universidade Estadual de Londrina, formado pelo artigo:
F.A. Guarnier, A. A. Suzukawa, A. N. C. Simão e R. Cecchini. Oxidative stress injury
in rat skeletal muscle with cachexia induced by Walker-256 tumor. FEBS Letters.
As formatações do artigo e das referências bibliográficas seguem as normas da revista.
2
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DEDICATÓRIA
Aos meus pais, por seu amor
incondicional, e por terem me
ensinado que existem valores muito
mais importantes do que os materiais.
A DEUS, que, com certeza, nesta
etapa e sempre, me acompanhou e me
permitiu fazer o que gosto.
Ao Fernando, que vem sendo o meu
maior companheiro em todas as
minhas caminhadas e decisões.
3
AGRADECIMENTOS
Em primeiro lugar, não ao meu orientador, mas ao meu amigo e mestre Prof. Dr. RUBENS
CECCHINI, que me ensinou os maiores valores que um pesquisador-ser humano pode possuir, me
ouviu chorar, me serve de exemplo, e que, acima de tudo, me ensinou a domar essa ansiedade...
À minha família, que me deu base e formação moral, apesar da distância, para sempre enfrentar
desafios, nunca me acomodar, e, acima de tudo, ser forte.
À amiga mais competente que eu poderia ter, A
NDRÉIA AKEMI SUZUKAWA, que chorou,
reclamou, riu, fez repique e tomou Clight comigo nestes dois anos, além de ter tornado esta
caminhada muito mais suave, claro.
Ao meu amigo Z
UI, que, além de técnico do laboratório, foi o meu braço direito, braço esquerdo,
meus dois pés, meu confidente, conselheiro, e tudo mais....
À Profa. Dra. ALESSANDRA CECCHINI, que me deu a oportunidade de atuar na minha área de
formação, e se tornou uma grande companheira de laboratório.
Ao Prof. Ms. JAIR TONON, e aos amigos de laboratório SOLANGE, KARINA, MARIA FERNANDA,
WANDERLEI, WANDER, CRISTIANE, LEILA, PATRÍCIA, PAULA, VERA, CELSO, e ALEXANDRE, que
me ajudaram a ter bom humor nos momentos mais complicados (que nem foram tantos assim).
Aos amigos da turma do mestrado, KAREN, CARLOS EDUARDO, LIGIANE, FABRINE, MARI, LEILA,
ANDRÉIA, e em especial, à HELEN, que dividiu comigo um dos melhores momentos deste curso.
Aos funcionários do Laboratório de pós-graduação (LPG/HU), pela paciência, disposição e
colaboração na realização de vários trabalhos do nosso laboratório, em especial ao Prof. Dr.
DÉCIO SABATTINI BARBOSA, que sempre se mostra à disposição.
Aos meus irmãos G
LÁUCIA e DANILO, por estarem sempre presentes. Em especial ao Danilo, que
além de ter sido sempre o meu companheiro, lavou a louça todas as vezes que eu pedi.
4
RESUMO
MATERIAIS E MÉTODOS─ Foram utilizados ratos Wistar machos de 200-250g, com água
e ração comercial (Nuvilab
®
) oferecidas ad libitum. Verificou-se o consumo de ração diariamente.
Os ratos foram divididos em dois grupos. O primeiro (n=6) recebeu inoculação subcutânea, no
flanco direito, de 0,5 mL de PBS (controle). O segundo grupo (n=18) recebeu implantação
subcutânea no flanco direito de uma supensão de 8,0 x 10
7
células tumorais em 0,5 mL de PBS.
Realizou-se também um controle paralelo, onde 6 animais inoculados com PBS receberam a
mesma quantidade de ração previamente ingerida pelos animais inoculados com tumor, durante
14 dias (pair-fed). Nos dias 5, 10 e 14, após a inoculação do tumor, os animais (6 por dia de
sacrifício) foram pesados, decapitados, e o tumor cuidadosamente dissecado e pesado.O músculo
gastrocnêmio esquerdo de cada animal foi rapidamente retirado para pesagem e armazenagem em
nitrogênio líquido, até o momento do uso. O índice de porcentagem de massa muscular/
INTRODUÇÃO─A caquexia é definida como uma síndrome de progressiva perda de
peso, anorexia, e persistente perda de massa magra. É característica freqüente em
pacientes portadores de câncer. A manifestação da caquexia é caracterizada por perda
maior que 10% da massa corporal total, e é responsável por cerca de 22% dos óbitos em
pacientes com doença maligna avançada. A perda maciça de massa muscular é
responsável pela maior parte da perda de peso total, e pode ocorrer independentemente da
diminuição na ingesta alimentar ou má absorção de nutrientes. Essa perda muscular
significativa é conseqüência do desequilíbrio entre síntese e degradação de proteínas, e
provoca astenia (perda de força muscular), vista ainda no estágio inicial da doença.
Recentemente, alguns estudos demonstraram o envolvimento da via proteolítica
ubiquitina-proteasoma na perda de massa muscular na caquexia. Algumas citocinas pró-
inflamatórias, produzidas por células tumorais, também têm demonstrado envolvimento
na patogênese da perda de massa muscular, como IL-1, IL-6, e TNF-α, que foi
originalmente chamada de “caquectina”. O estresse oxidativo é definido como uma
produção excessiva das espécies reativas de oxigênio (ERO) que não podem ser
neutralizadas por defesas celulares antioxidantes. Recentemente, o estresse oxidativo tem
conseguido especial atenção por seu possível envolvimento na caquexia do câncer. Alguns
estudos têm demonstrado que o estresse oxidativo moderado pode aumentar a degradação
protéica através do aumento na expressão dos componentes da via ubiquitina-
proteassoma. Entre as diferentes modificações induzidas pelas ERO em resíduos de
aminoácidos, a carbonilação protéica consiste em um dos melhores marcadores de lesão
de proteínas em vários estados patológicos. A peroxidação lipídica representa um dos
mais significativos processos que precedem a degeneração celular e necrose, sendo
extensivamente representada pela produção de malondialdeído (MDA). Estudos mais
recentes utilizam um método mais sensível para detecção de lipoperoxidação, por
quimiluminescência.
Considerando todas essas informações, e também a existência de poucos dados na
literatura a respeito de lesões por estresse oxidativo, em músculo esquelético na caquexia
do câncer, esse trabalho se propôs esclarecer o envolvimento do estresse oxidativo no
desenvolvimento da caquexia relacionada ao câncer, em músculo esquelético de ratos com
a forma subcutânea do tumor de Walker-256, um modelo experimental que vem sendo
utilizado para indução de caquexia experimental. Para esse propósito, foram explorados
alguns dos mais importantes sistemas antioxidantes e a injúria às proteínas e aos
lipoperóxidos de membrana, em diferentes tempos da progressão do tumor.
5
RESULTADOS― Como principais resultados, obtivemos:
1) A implantação do tumor de Walker-256 levou à diminuição progressiva do peso
corporal no 5º (10,55% de perda em relação ao controle) e 10º(14,27%) dias, que foi
acompanhada pela progressiva perda de massa do músculo gastrocnêmio (2,9%, 12,3%,
e 15,8% no 5º, 10º e 14º dias, respectivamente). Observou-se também progressivo
aumento da massa tumoral e diminuição na ingesta alimentar dos animais inoculados
com células tumorais, quando comparados ao grupo controle. Os animais que receberam
a mesma quantidade de ração consumida pelos animais inoculados com o tumor
apresentaram, em 14 dias (10.1 ± 1.55%)a mesma diminuição encontrada no grupo 5
dias, quando comparado ao controle..
2) Os índices de massa muscular/massa corporal total observados foram: um aumento de
0,56 no grupo pair-fed, e diminuições de 0,27 no grupo 5º dia, 0,86 no grupo 10º dia, e
0,81 no grupo 14º dia.
3)A lipoperoxidação, avaliada pela formação de TBARS, mostrou um aumento
significativo no 10º dia, chegando a aproximadamente 2,5 vezes os valores encontrados
no grupo controle. Os níveis de TBARS retornaram aos do grupo controle no 14º dia.
4)A QL estimulada por t-butil hidroperóxido mostrou aumento significativo (p<0,001)
em todos os grupos experimentais avaliados (5º, 10º e 14º dias), mostrando maior
evidência no 5º dia após inoculação (pico de 6,23 Unidades Relativas de Luz) das
células tumorais. Os valores do V
0
mostraram-se progressivamente aumentados até o 14º
dia: 58 x 10
-3
no grupo controle, e 62,5 x 10
-3
; 92,5 x 10
-3
, e 103,10
-3
Unidades Reativas
de Luz por minuto, respectivamente).
5)Constatou-se uma diminuição significativa na concentração de TRAP, já no 5º dia
(0,357 ± 0,03 μM de trolóx) após a implantação do tumor, que reverteu-se completa e
progressivamente no 10º dia (1,277 ± 0,08 μM de trolóx). No 14º, ultrapassaram (2,186
± 0,19 μM de trolóx), de forma significativa, os níveis do grupo controle (1,217 ± 0,13
μ
M de trolóx
)
.
porcentagem de massa corporal total perdida também foi calculado, para que se pudesse ter uma
clara idéia da quantidade de massa muscular perdida, em relação ao total.
Para os ensaios de Quimiluminescência induzida por hidroperóxido de t-butil (QL) e Teste das
Substâncias Reativas ao Ácido Tiobarbitúrico (TBARS), os músculos foram homogeneizados
em banho de gêlo [10 mg de tecido/mL de tampão Fosfato de Potássio Monobásico
(KH
2
PO
4
/K
2
HPO
4
)
,
120 mM KCl, pH 7.4]. Para os ensaios de atividade da Superóxido
Dismutase (SOD), Capacidade Antioxidante Total (TRAP), e para atividade do sistema
glutationa, utilizou-se do sobrenadante de um homogenato 50 mg/mL do mesmo tampão,
centrifugado a 11000 x g por 15 minutos, a 4º C. Para obtenção de proteínas carboniladas,
utilizou-se homogenato total 50mg/mL.
A avaliação da lipoperoxidação de membranas celulares de músculo esquelético foi realizada
através do teste das substâncias reativas ao ácido tiobarbitúrico (TBARs) e Quimiluminescência
induzida por t-butil hidroperóxido (QL). A atividade da superóxido dismutase (SOD) e a
quantificação das glutationas reduzida e oxidada, assim como o índice de estresse, foram obtidos
através de procedimentos padronizados. A Capacidade Antioxidante Total (TRAP) foi avalida
por luminescência, utilizando trolóx como padrão. Para detecção de proteínas carboniladas,
utilizou-se o método colorimétrico, através da reação com a Dinitrofenilhidrazina (DNFH).
Os resultados representam a média e erro padrão de 6 animais por grupo. A análise de
significância foi realizada por teste t de Student, para dados não-pareados. Para os resultados de
QL, utilizou-se teste t de Student para dados pareados. Consideraram-se significativos os
resultados quando p< 0,05.
6)As concentrações de glutationa reduzida (GSH) apresentaram queda progressiva até o
14º dia (4,040 ± 0,1 no grupo controle, 1,965 ± 0,2 no 5º, 1,490 ± 0,1 no 10º, 1,267 ±
0,05 μM/mg de proteína no 14º dias), o que não acarretou em aumento nas
concentrações
de GSSG (0,345 ± 0,04 no grupo controle, e 0,310 ± 0,2; 0,172 ± 0,02; e 0,378 ± 0,03
μM/mg de proteína respectivamente Além disso, o índice de estresse revelou-se da
seguinte forma: 8,71 ± 2,7 no grupo controle, 18,98 ± 1,9 no 5º dia, 11,94 ± 1,0 no 10º
dia, e 20,03 ± 2,5 no 14º dia.
7)A atividade da SOD mostrou-se significativamente aumentada no 5º (0,240 ± 0,03 U
SOD/mg de proteína) e 10º (0,195 ± 0,01 U SOD/mg de proteína) dias após a
implantação do tumor, recuperando a atividade dos níveis controle (0,131 ± 0,006 U
SOD/mg de proeína) no 14º dia (0,101 ± 0,01 U SOD/mg de proteína).
8)Os níveis de proteínas carboniladas mostraram um aumento progressivo (2,176 ± 0,23
proteínas carbonílicas/mg de proteínas totais no grupo controle, 2,608 ± 0,19 no 5º dia),
atingindo o seu máximo no 10º dia (3,046 ± 0,19), e retornando aos níveis encontrados
no grupo controle no 14º dia (2,478 ± 0,26 proteínas carbonílicas/mg de proteínas
totais).
CONCLUSÕES— Este estudo, mostra claras evidências sobre a existência de uma
associação entre a caquexia induzida por câncer experimental, lipoperoxidação e ataque
maciço a proteínas. Os principais mecanismos antioxidantes foram explorados e
apontados, o que provavelmente indica lesão durante a progressão da doença.
DISCUSSÃO— A) A presença do tumor causou perda progressiva de massa total nos
animais, com a máxima relação perda muscular/corporal sendo atingida no 10º dia.
Estes dados coincidem com os dias em que foram apresentados os maiores níveis de
MDA, indicando que a peroxidação lipídica acontece concomitantemente à diminuição
da massa muscular. Como os grupos pair-fed não apresentaram perda significativa de
massa muscular e corporal em relação ao controle, estes resultados provavelmente
devem-se à ação sistêmica do tumor.
B) A análise total da curva de QL revelou que os maiores níveis de emissão foram
alcançados já no 5º dia após a inoculação, se mantendo significativos até o 14º dia. Estes
resultados apontam para um rápido consumo das defesas antioxidantes não-solúveis
antes que níveis significativos de lipoperóxidos se estabeleçam, o que foi reforçado pela
determinação dos valores de V
0
, que se elevaram no 10º dia após a implantação do
tumor.
C) Os níveis de proteínas carboniladas dos animais com caquexia induzida por tumor
apontaram para o mesmo padrão de lesão mostrado nos testes de TBARS e QL, além de
coincidir com a maior relação entre perda de massa muscular e massa corporal.
Considerando o fato deradicais livres reagem prontamente com proteínas, e que, como
consequência, modificações de proteínas podem ocorrer através da reação de radicais
secundários com aldeídos de baixo peso molecular, podemos então sugerir que o
estresse oxidativo possa estar envolvido na regulação do mecanismo proteolítico.
D) O padrão dos três sistemas antioxidantes investigados no músculo de ratos caquéticos
foi significativamente diferente dos músculos controle. Estes resultados apontam para
mobilização do sistema antioxidante, contra a lesão, num estado precoce do avanço da
síndrome da caquexia.
7
OXIDATIVE STRESS INJURY IN RAT SKELETAL MUSCLE WITH CACHEXIA
INDUCED BY SOLID WALKER-256 TUMOR.
Flávia Alessandra Guarnier, Andréia Akemi Suzukawa, Andréa Name Colado Simão, and
Rubens Cecchini.
Physiopathology Laboratory of Free Radicals, Universidade Estadual de Londrina,
86051990 Londrina (Brazil)
Mail adress:
Rubens Cecchini
Physiopatology Laboratory of Free Radicals
Department of Pathology Sciences
Universidade Estadual de Londrina (UEL)
E-mail: [email protected]
Fax (043)33714267
86051-990 Londrina, Brazil
Abreviations: Tumor Necrosis Factor – TNF; Malondialdehyde – MDA; Reactive Substances of
Thiobarbituric Acid – TBARS; Chemiluminescence – CL; Total Antioxidant Capacity – TRAP; Relative
Light Units – RLU; Reduced Glutathione – GSH; Oxidized Glutathione – GSSG; Stress Index - SI;
Superoxide Desmutase— SOD, Reactive Oxygen Species – ROS, RNS – Rective Nitrogen Species.
8
ABSTRACT
Cachexia is a wasting syndrome, characterized by progressive weight loss, generally
asssociated to the cancer and other inflammatory diseases. Studies revealed increases on
TBARS and CuZn SOD on hypothalamus of rats with cachexia induced by Walker-256
tumour. Other studies showed that moderate oxidative stress in skeletal muscle of tumour
bearing rats enhanced protein degradation by the ubiquitin-proteasome proteolytic pathway.
This work wanted to clarify the involvement of oxidative stress on skeletal muscle of rats
with Walker-256 carcinosarcoma. For this purpose, male Wistar rats were inoculated
subcutaneously with a suspension of tumour cells. On days 5, 10 and 14 after tumour
implantation, the animals were weighed, killed by decapitation, and the tumour carefully
excised and weighed. The gastrocnemius muscle was rapidly excised, weighed and stored
at liquid nitrogen until use. Then, the muscles were homogenized in cold buffer and
oxidative stress evaluated by the tiobarbituric acid reactive substances (TBARS),
chemiluminescence induced by t-butyl (CL), and quantification of antioxidants defenses
(TRAP, SOD, Glutathione). In addition, the oxidation of proteins was evaluated by
amounts of carbonyl proteins. Our results revealed a progressive loss of body weight on 5
th
and 10
th
days after tumour implantation, what was accompanied by muscle mass loss. The
levels of TBARS enhanced 2.5 times on 10
th
day. The CL increased significantly (p<0.001)
in all experimental groups, showing proeminent elevation of lipid peroxidation on 5
th
day.
The TRAP values decreased on 5
th
day, returning to control levels on 10
th
day. The Stress
Index, calculated through total and oxidized glutathione concentrations, showed 2 and 3
times of enhacement on days 5 and 14, respectivelly. The SOD activity increased from
0.195 ± 0.01 USOD/mg of protein on 5
th
day, to 0.240 ± 0.03 USOD/mg of protein on 10
th
day. Carbonyl proteins increased either, although this enhacement has appeared only on
10
th
day, declining on day 14. The results revealed the involvement of oxidative stress on
weight loss process of skeletal muscle with cachexia induced by Walker-256 tumour. In
addition, the enhancement of carbonyl proteins could feed the proteolysis process and
consequent promote reduction of muscle mass.
Keywords: cachexia, Walker-256, skeletal muscle, oxidative stress, lipid peroxidation,
chemiluminescence.
9
1.INTRODUCTION
Cachexia is a complex and multifatorial syndrome, responsible for 22% of patients
death [1]. About 30% of body mass loss weight is invariably fatal [2]. Muscle wasting
accounts for the majority of the muscle loss, which may occur independently on the
decrease of food intake or malabsorption of nutrients [3]. Thus, asthenia (lack of muscular
strength) reflects the important muscle waste that takes place in the cachectic cancer
patient, and it is one of the most relevant characteristics generally associated to this
syndrome [4].
Loss of skeletal muscle in patients and animals is a consequence of protein synthesis
and degradation inbalance, as indicated by a variety of metabolic alterations [5]. During the
past decade, several studies have been performed in order to clarify the contribution of
different proteolytic pathways to muscle wasting, and the mechanisms responsible for their
activation and regulation. Of the proteolytic pathways contained in the skeletal muscle, the
lysosomal and the protesomal systems can operate a degradative proteolysis, while the
calcium-dependent (like calpains) and the caspase systems only operate limited or partial
proteolysis [6]. The ubiquitin-proteasome system, which is crucially involved in the
degradation of regulatory and abnormal cellular proteins, is believed to provide most, at
least, of the proteolytic activity required for the degradation of muscle ptotein [7]. The
expression of genes pertaining to this system and the amount of ubiquitin-protein
conjugates are increased in atrophying muscles in cancer conditions [6]. Further evidences
show that muscle proteasomal activity, as assayed with peptide substrates, is enhanced in
experimental models of cancer cachexia [8], as well as in some cancer patients [9]. Several
10
cytokines have been implicated in the pathogenesis of muscle wasting, such as TNF-α, IL-1
e IL-6. TNF-α, that was first called “caquectin”, and IL-1, bind to its receptors and induce
the activation of the NF-κB transcription factors [10]. It has been recently demonstrated
recently that activation of the NF-κB transcription pathway, activated by cachectic factors
as TNF-α, is sufficient to induce skeletal muscle atrophy, and that occurs in part via NF-κB
[11]. The involvement of oxidative stress on the ubiquitin-proteasome proteolytic pathway
has been suggested [12].
Oxidative stress is defined as an imbalance between production of reactive
oxygen/nitrogen species, and antioxidant defense. Since this state can cause damage to all
types of biomolecule, including, proteins and lipids [13], it has gained attention for its
possible involvement on muscle damage of cancer cachexia. Gomes-Marcondes and
Tisdale [12] showed that muscle wasting in cancer cachexia is asociated with increased
levels of malondialdehyde in gastrocnemius muscles, and that mild input in ROS
generation can increase protein degradation in skeletal muscle by causing a greater
expression of the major components of the ubiquitin-proteasome system [14], as proteins
are one of the major targets of oxidative stress-derived effects in tissues [1]. Similarly,
Buck and Chojkier [14] demonstrated that muscle wasting and dedifferentiation could be
prevented by treatment with α-tocopherol or BW755c antioxidants, reverting the cachectic
status caused by TNF-α. Freitas et al [15] revealed increased indices of lipid peroxidation
and antioxidant enzymatic activity in brain regions of rats bearing solid tumor. On the other
hand, muscles of rats bearing an ascitic form of tumor presented no significative differences
on antioxidant enzymatic activity, after tumor implantation [1]. Some studies have pointed
out to the involvement of glutathione system through regulation of protein ubiquitinylation
11
[16-18], and of hydrogen peroxide through the phosphorilation of Iκb, a part of NFκB that
feeds the proteolytic pathway [10, 12].
The most used method to demonstrate lipid peroxidation levels is the production of
MDA [1, 12]. In early work, the measurement of oxidative damage on lipids in skeletal
muscle by a very sensitive chemiluminescence procedure, was shown [19-21]. Proteins
Among the different modifications induced by ROS in amino acid residues, protein
carbonylation consists one of the best characterized markers of protein damage in several
conditions and disease states [1, 22].
Considering all this information, and since there are poor evidences about the
oxidative stress damage in skeletal muscle of cancer cachexia, we proposed to clearify the
involvement of oxidative stress in the development of cancer-related cachexia in muscles of
rats bearing the subcutaneous form of Walker-256 carcinosarcoma, a tumor that has been
extensively used as an experimental model to induce cachexia in rats [23-25]. For this
purpose we explored muscle levels of several antioxidant, protein and lipoperoxidative
injury in three different time courses of tumor progression.
12
2. MATERIAL AND METHODS
2.1. Animals
Adult male Wistar rats, obtained from the Animal House of the Biological Sciences
Center at the Universidade Estadual de Londrina, weighing 200-250g, were used
(n=6/group). The animals had water and comercial food (Nuvilab CR1, Nuvital Nutrients
Ltda., Curitiba, Brazil) ad libitum. The food intake was measured daily. All animals were
carefully monitored and maintained in accordance with ethical recommendations for animal
experimentation.
2.2. Tumor inoculation
Rats were divided into two groups, named controls and tumor hosts. The former
received 0.5 mL of a PBS solution injection and the latter received a Walker-256 cell
suspension (8,0 x 10
7
cells in 0,5mL of PBS), subcutaneously injected on the right flank.
Tumor cells were maintained in our laboratory as an ascitic intraperitoneal tumor, after 1
week of the injection of 2.0 x 10
6
cells /0.5 mL of PBS.
A food intake control group was carried out, where 6 animals innoculated with PBS
were fed with the same amounts of food consumed by tumour group, during 14 days (pair-
fed). On days 5, 10 and 14 after subcutaneous tumour implantation, the animals were
weighed, killed by decapitation, and tumor was carefully excised and weighed. The
cachectic index was determined by the following formula and should be above 10% to
characterize cachexia.
13
% loss of body weight = [ibm – fbm + (tw) + gbm]
X 100%,
(ibm + gbm)
where, ibm = inicial body mass of the tumour bearing animal, fbm = final body mass of the
tumour bearing animal, tw = tumor weight, and gbm = mean of gain of control group body
mass. The ratio % of muscle mass loss / % of total body mass loss was calculated in order
to obtain a pattern of general waste.
The contralateral gastrocnemius of tumour bearing animals were rapidly excised,
weighed, and stored at liquid nitrogen until use (at most 60 days of storage). The animals of
the control group that received PBS subcutaneous injection were treated at the same
manner, and compared with experimental groups.
2.3. Tissue prepare
Muscles were placed on ice and homogenized five times for 30s periods cycles with
60s intervals in an Ultraturrax homogenizer, containing 10 mg of tissue/mL of 30mM
KH
2
PO
4
/K
2
HPO
4
buffer and 120 mM KCl, at pH 7.4. This homogenate was used for the
tert-butil hydroperoxide-stimulated chemiluminescence, and TBARS assays. The
supernatant of the homogenate was obtained by centrifugation at 11,000 x g for 15 min at
4ºC, from a homogenate containing 50 mg of tissue/mL of the same buffer, and used for the
TRAP, glutathione, and SOD assays. For total protein carbonylation, tissues were specially
treated, according to Reznick and Parker [26], with some adaptations, as described later.
2.4. Determination of Thiobarbituric Acid Reactive Substances (TBARS)
14
The lipoperoxidation of muscle cells of control, 5, 10 and 14 days of tumor
homogenates were determined by TBARS reaction, where MDA levels were measured and
the resulta were expressed in nanomoles MDA/g tissue, as described by Oliveira and
Cecchini [27].
2.5. Measurement of tert-butil hydroperoxide-initiated chemiluminescence of muscle
homogenates
Reaction mixtures were placed in 2mL luminescence tubes, containing the
following at the respective final concentration: total muscle homogenate from contralateral
gastrocnemius of tumour bearing rats (10 mg/mL), 30 mM KH
2
PO
4
/K
2
HPO
4
buffer (pH
7,4, 120 mM KCl), and 3 mM tert-butil hydroperoxide, in a total volume of 1 mL. The tert-
butil hydroperoxide-initiated chemiluminescence (CL) reaction was measured in a TD/20
20 luminometer (Turner Designs), with a response range from 300-650 nm. The tubes were
kept in the dark up to the moment of assay, which was carried out in a dark room at
approximately 28ºC [28, 29]. Results are expressed in Relative Light Units/g tissue (RLU/g
tissue). The entire curve was used as an indicator of lipid peroxidation. V
0
values were
obtained by linear regression of the ascending part of the CL curve.
2.6. Measurement of the total antioxidant capacity of muscle (TRAP)
Total antioxidant capacity of the muscle homogenate was measured by CL, in a
reaction medium containing 20μM 2,2-azo-bis-(2-amidinopropano) and 200 μM luminol.
The addition of 70 μL of each supernatant (control, 5, 10 and 14 days - 50 mg/mL)
decreased the CL to basal levels for a period (induction time) proportional to the
15
antioxidant content of the sample until reaching the CL regeneration level [30, 31]. The
system was calibrated with equal concentration of trolox.
2.7. SOD activity
The oxidative stress interference on the SOD activity was determined according to
Marklund and Marklund (1974) [32], based on the inhibition of pirogalol autoxidation in
acquous solution. SOD inhibits pirogalol oxidation by catalyzing O
2
-
to H
2
O dismutation.
This oxidation is accompanied by yellow color formation in the reaction medium,
monitored at 420 nm. In the reaction, crescent volumes (125, 150, 175 and 200 μL) of
contralateral gastrocnemius supernatant are diluted in 100μL of TRIS buffer 1M HCl 5mM
EDTA, pH 8.0, and, after that, 15μL of pirogalol is added. The reaction was monitored for
5 minutes, and the absorbance, in the beginning and in the end of the assay, was registered.
The auto-oxidation of pirogalol was used as control. The amount of SOD that is able to
inhibit pirogalol autoxidation in 50%, is defined as the enzymatic activity unit (U). Final
results were expressed in U SOD/ mL
.mg protein
–1
. min
–1
2.8. Glutathione Assay
The levels of reduced glutathione (GSH) were determined by titration with 5,5’-
dithio-bis (2-nitrobenzoic acid), evidenced by a yellow color formation. Oxidized
glutathione (GSSG) was determined at the same manner, in the supernatant previously
incubated with 4-vinylpyridine for 60 min at room temperature according to the method
proposed by Tietze (1969) [33]. Volumes of supernatant were adjusted for the assay with
muscle homogenate, containing 50mg/mL. The results were expressed in μmol/mg protein.
The stress index was calculated, by the equation [(GSSG/GSH-GSSG) x 100].
16
2.9. Carbonyl proteins content
For access carbonyl proteins content, we used the method described by Reznick and
Parker (1994) [26], with adaptations. About 200 mg of contralateral gastrocnemius muscles
from control and tumor animals were placed on homogenization glass tubes containing 4
mL of homogenizing buffer (50 mM phosphate buffer, 1 mM EDTA, pH 7.4). Tissues
samples were finally homogenized and incubated for 15 min at room temperature. Thus,
samples were centrifuged at 3,000 x g for 10 min at room temperature to remove debris,
and 1 mL of each extracted protein was placed on glass tubes. About 4 mL of 2,4-
Dinitrophenylhydrazine (DNPH) diluted in 2.5 M HCl solution was added in each tube and
left for 1 h of incubation at room temperature, vortexed every 15 min. Then, samples were
washed with 5 mL of 20% TCA (w/v) solution and centrifuged for 10 min to collect the
protein precipitates. Another wash was performed using 10% TCA, and protein pellets were
broken mechanically. Finally, the pellets were washed 3 times with 4 mL of ethanol-
ethylacetate (1:1, v/v) to remove free DNPH and lipid contaminants. The final precipitates
are dissolved in 2 mL of 0.6 M guanidine hydrocloride solution and any insoluble materials
are removed by additional centrifugation. Carbonyl content was calculated by obtaining the
peak of absorbance on a spectra at 355-390 nm of the DNPH-treated samples, against
samples treated only with 2.5 M HCl. To calculate the concentration of carbonyls, we used
the described formula: C = Abs(355-390) x 45.45 nmol/mL, where C is the concentration
of DNPH/mL, and 45.45 its absortion coefficient [26]. The procedures were made on ice
bath until TCA wash. Results were expressed in nanomoles of carbonyls. mL
-1
.mg total
protein
–1
.
17
2.10. Protein concentration
Protein was determined by the method of Lowry et al [34], modified by Miller [35],
except on protein carbonyl contents, when we used a spectra at 280 nm of each sample to
determine total protein content. Both methods used bovine serum albumin (BSA) as
standard. A calibration curve to determine the concentration of BSA was made.
2.11. Statistical analysis
The results are shown as means ± SEM of six animals. Data were evaluated using a
non-paired Student’s t test. Correlation analysis was used to determine V
0
. p < 0,05 was
considered significant.
18
3. RESULTS
3.1. Characterization of cachexia
Table 1 shows the characterization of cachexia on tumor-bearing animals. Walker-
256 tumor leads to a progressive decrease in body weight during the course of 14 days.
General body weight loss was accompanied by loss of contralateral gastrocnemius weight
(2.9, 12.35 and 15.88% when compared to control). Pair fed group did not present
significant differences in muscle and body weight, when compared to control group. Figure
1 demonstrates the rate of muscle mass loss compared with total body mass loss. On 10
th
day, muscle (12.33 ± 3.00%) represented the major part of general body loss (13.1 ±
1.75%), which was mantained on day 14 (15.88 ± 3.36% and 20.01 ± 3.86%, respectively).
We also observed progressive increase in tumor weight and decrease in food intake at the
same period, when compared with control group.
3.2. Lipoperoxidative damage
Lipoperoxidation in cell membranes of skeletal muscle was determined by the
progressive formation of TBARS (Figure 2) in the reaction mixture, showing a significant
increase of approximately 2.5 times on day 10 after tumor inoculation when compared with
control group (from 0.391 ± 0.022 on control, to 0.900 ± 0.1 nanomoles MDA/g tissue on
Day 10). However, when TBARS was measured in day 14, the levels returned to the ones
observed on the control groups (0.468 ± 0.09 nanomoles MDA/g tissue), representing no
significant differences.
Tert-butyl hydroperoxide-initiated chemiluminescence was used to analyze the
integrity of non-enzymatic antioxidant defenses, and the levels of lipid peroxides in muscle
19
cells of animals exposed to tumor action. This assay indicates that the increase in CL is
closely related to the oxidative stress previously suffered by the tissue, inducing the
consumption of antioxidant defenses such as vitamin E and the formation of lipoperoxides
resulting in an increase in photon emission [27, 30, 36]. Figure 3 shows a significant
increase of total CL on all days of inoculation (p < 0.001 for all curves, when compared to
control). We found, however, increased values of V
0
. This value represents the initial
velocity of the reaction, pointing to the concentration of lipoperoxides present on tissue. It
was observed to be time-dependent, being more representative on day 10 (58 x 10
-3
URL/min on control group;
3
62.5 x 10
-3
URL/min on 5
th
day; 92.5 x 10
-3
. URL/min on 10
th
day; and 103 x 10
-3
URL/min on 14
th
day after tumour implantation)
3.3. Total antioxidant capacity of the muscle
Figure 4 show the values of total antioxidant capacity (TRAP) of contralateral
gastrocnemius muscle of the animals. Control values were 1.217 ± 0.13 μM trolox
equivalents; a significant decrease in antioxidant capacity was observed on day 5
(p<0.001), which was completely and progressive reversed on days 10 (1.50 ± 0.1 μM
trolox) and 14, which showed to be higher than the control levels.
3.3. Antioxidant enzymes
GSH levels were progressive and significantly decreased on day 5, 10 and 14 (51%,
63%, and 68%, respectively, of reduction, when compared to control), which was not
accompanied by the increase on GSSG levels. GSSG tended to decrease already on day 5
(10% of reduction), achieving significant difference in day 10 (50% of reduction, p< 0.05).
On day 14, GSSG returned to level similar to control, but not presented significant
20
difference when compared to control. Yet, stress index (SI), that represents cellular
oxidative stress, has demonstrated significant increased differences, when compared to
control, on days 5 and 14 (increasing about 54 and 56%, respectively). Absolute values (in
means ± SE) can be seen on Table 2.
Figure 5 shows that the activity of superoxide dismutase was significantly
enhanced on days 5 and 10 (from 0.131 ± 0.01 U SOD/mL/mg protein/min on control
group, to 0.240 ± 0.03 on day 5, and 0.196 ± 0.01 on day 10), recovering control levels on
day 14 (0.108 ± 0.08 U SOD/mL/mg protein/min), showing again activity against oxidative
damage on days 5 and 10.
3.4. Protein Carbonylation
For the carbonylated proteins, it is evidenced that only the day 10 there is a
significant increase (Figure 6) on carbonyl proteins levels, (approximately 33,3% on day
10), which returns to control levels on day 14. Reflecting, at this point, not only damages to
muscle cell lipids membranes, but also to proteins.
21
4. DISCUSSION
To investigate the role of oxidative stress on gastrocnemius muscle induced by
cancer cachexia, we chose an experimental model that has notable cachectic response.
Walker-256 tumor is a rat tumor that grows exponentially, becoming ulcerative when
implanted subcutaneously. Its growth promotes a survival time about 14±1 days with
concomitant reduction of food intake [8, 37]. In addition, the presence of tumor causes
rapid and progressive loss of body weight and tissue waste, particularly in skeletal muscle
[38]. Body and muscle wasting occurred 5 days after tumour inoculation, where cancer
development was evident only after 10 days. The muscle/body rate was 0.27 upon the day 5
and increased to 0.86 and 0.81 on the days 10 and 14 respectively. These data suggest a
marked effect of cancer development on muscle damage after 10 days of inoculation. The
control group which was carried out with food restriction (pair fed) did not induce any
significant body weight loss and muscle wasting. Studies using TNF or Walker-256 tumor
inducing cachexia, revealed reduction in animal food intake [1,14].
Previous studies have suggested that ROS could have a central role in muscle
wasting [1, 14, 15, 23]. Decreased weight and muscle wasting were accompanied by
increased MDA levels in skeletal muscle of rats injected with TNFα. These parameters
were reverted by α-tocopherol treatment [14]. MDA and 4-hydroxy-2-nonenal (HNE)
adducts were found to be increased in gastrocnemius of animals bearing the ascitic form of
AH-130 Yoshida hepatoma, when compared to controls [1]. Freitas et al [15], using rats
bearing Walker-256 solid tumor, demonstrated enhanced TBARs in some brain regions
after 14 days of subcutaneous implantation. It has been demonstrated that twenty days after
22
cachectic tumor MAC16 transplantation in mice or in vitro C
2
C
12
myotubes treatment with
either H
2
O
2
or hydroxyl radical generating system, caused a significant rise in the MDA
content [12]. In addition, these authors suggested that mild oxidative stress increases
protein degradation in skeletal muscle by causing up regulation in the ubiquitin proteasome
proteolytic pathway. Therefore, supported by evidences obtained in the present work, we
suggest that muscle waste is a result of multifatorial biochemichal alterations, in which
oxidative stress has an important involvement. First, by oxidative stress as a mediator of
tissue injury, including protein oxidation, and second, by as a modulator of muscle wasting
process. To understand these mechanisms, the oxidative stress injury in muscle lipid was
evaluated by a very sensitive chemiluminescence method besides MDA and carbonylated
protein measurements. Additionally, different antioxidant systems were measured in
several time after tumor implantation. Our results showed an incresing tendency in the
MDA levels on 5
th
day after tumor implantation (figure 2), reaching higher concentration
on 10
th
day, declining afterwards to control levels on 14
th
day. These data suggest that lipid
peroxidation is a continuing event during the muscle wasting process, and are coincident
with the maximal muscle wasting/body mass rate, at the day 10 after inoculation. Besides
of a tert-butil hydroperoxide-initiated chemiluminescence curve measurements, we carried
out a kinetic analysis of the ascending part of the CL curves as an indicator of membrane
lipid peroxidation (figure 3) [27, 28, 36]. Zamburlini et al [39] used purified lipid
hydroperoxides or plasma LDL lipid hydroperoxides and found that emission obtained was
proportional to the lipid hydroperoxide content of the sample. The relationship between
chemiluminescence and tissue damage has been previously demonstrated [20, 21, 27, 36,
40]. In the present study, using contralateral gastrocnemius from rats hindlimbs subjected to
tumor implantation, the total analysis of the CL curve revealed that the higher emission
23
levels was achieved as early as 5 days after inoculation, maintaining significantly high until
the day 14. These results show a fast consumption of non soluble antioxidant defense
before significant levels of lipid peroxides levels have been attained. Additionally, the V
0
values were elevated only in the 10
th
after the tumor implantation. (figure 3). These results
reveal a massive free radical attack mainly on the cells membrane, promoting increased
levels of tissue lipid peroxides, which agree with the maximal MDA levels and also, with
wasting muscle/body mass rate found on the 10
th
day. Considering that: (1) the fact that
proteins may be attacked whenever free radicals are generated, and, as a consequence,
oxidative modification of proteins may occur by reaction with diverse primary radicals as
•OH or secondary radicals as alkylperoxyl or low molecular aldehydes as MDA and HNE
[41-43] and (2) that, the increased susceptibility of oxidized proteins to undergo proteolytic
degradation [44, 45], we decided to detect whether carbonyl groups were increased in the
cachectic muscles. Total protein carbonyl group showed the same profile of MDA and CL
curve during the 14 days of tumor development. The more strike similarity occurs in the
10
th
day after the tumor implantation, showing higher values for all parameters analyzed.
Since our results demonstrated increased levels of carbonylated proteins on day 10 with the
concomitant increase on MDA levels, higher V
0
and wasting muscle values we can suggest
that besides of clear participation in tissue injury, the oxidative stress should also be
involved in the regulation of the proteolytic system that results in accelerated muscle
proteolysis, which is the tissue that defines the cachectic state and considered to be the first
cells that are subjected to metabolic alterations, specially by the activation of ubiquitin-
protesome pathway, possibly through free radical production, which in turns produces more
free radical, originating a vicious cycle.
24
The profile of the three antioxidants systems investigated in the muscle of cachectic
rats was significantly different with respect to control muscle. Thus, SOD and the stress
index raised significantly 5 days after tumor inoculation. On the same time, there were a
marked reduction in the total antioxidant capacity and GSH levels. The stress index is
reduced in the day 10, although SOD activity remains significantly high. The TRAP
returned to control levels in this time. The higher levels of antioxidant observed at the 5
th
day with decrease to 10
th
day of implantation agree with the onset of wasting antioxidant
defense before oxidative injury has been established. Augmented SOD was previously
found in brain of rats bearing Walker-256 tumor 14 days after tumor inoculation [15].
Similarly, Lawler et al. [46] found increased of SOD activity in soleus muscle subjected to
immobilization for 28 days.
In conclusion, this study provides important evidences of the existence of a clear
association between experimental cancer-induced cachexia in skeletal muscle, lipid
peroxidative, and massive protein damages. The levels of the major antioxidant
mechanisms were explored, showing activity and consumption in all of them. This
indicates that the damage has different patterns of lesion during disease progression. Cancer
cachexia is a severe debilitating disorder for which
there are currently few therapeutic
options. Over the past few years, basic science
advances have begun to reveal the role of
oxidative stress on the progression of cancer cachexia, and the knowledge of this pathway
gives an insight to the improvement of therapeutics strategies. Further studies are needed to
clarify and define the role of ROS and its part on this syndrome and, fortunately, leading to
new treatment strategies, possibly involving modulation of the effects of ROS molecules on
host metabolism.
25
5. ACKNOWLEDGEMENTS
Grant support was provided by Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior – CAPES. We thank J. A. Vargas of the Department of Pathological
Sciences – Universidade Estadual de Londrina, for excellent technical assistance.
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33
Table 1. Progressive body and muscle weight loss in tumor induced cachexia.
Control Pair-fed Day 5 Day 10 Day 14
Food Intake (% of mean)*
-
- 11.77 28.87 45.02
Tumour weight (mg)
- -
3.1 ± 0.7 14.58 ± 0.9 25.26 ± 2.1
Loss of body weight (% )*
-
10.1 ± 1.55 10.55 14.27 19.49
Gastrocnemius weight (mg)
%
1012.5 ± 28.9
-
1074.17 ± 46.36
+5.74
983.33 ± 5.4
-2.9
887.5 ± 74.5
-12.3
851.67 ± 2.1
-15.9
% muscle mass loss/%body
mass loss ratio
-
-0.56
0.27
0.86
0.81
* Percent of reduction compared to control (100%). Groups represent number of days after
subcoutaneous injection of 8.0 x10
7
tumor cells. Each group consisted of 6 animals. Controls
received an injection of 0.5 mL of PBS only.Values are expressed as mean ± SE or in % when
specified.
.
34
Pair-fed Day5 Day10 Day 14
0
2
4
6
8
10
12
14
16
18
20
22
24
weight loss (%)
Muscle weight
Body weight
Pair-fed Day 5
Day 10
Day 14
Figure 1. Relation between muscle mass loss (solid barr) and body mass loss
(striped barr) when compared with control groups (100%) on the tumor progression
of rats implanted with the solid form of Walker-256 tumor (5, 10 and 14 days after
innoculation). Both barrs starts on graph basis.
35
Controle 5º dia 10º dia 14º dia
0,0
0,2
0,4
0,6
0,8
1,0
#
**
*
MDA (nmoles/ g tissue)
Control
Day 5
Day 10
Day 14
Figure 2. TBARS levels in contralateral gastrocnemius muscle homogenates
of rats inoculated with Walker-256 tumor cells. The values above are
expressed as mean ± SE of 6 different animals. * p< 0.05, when compared with
Control Group;
**
p<0.05, when compared with Day 5; and
#
p<0.05, when
related with Day 10.
36
0 20406080
400
450
500
550
600
650
700
URL. g tissue
-1
Time
(in minutes)
Control
Day5
Day10
Day14
*
*
*
Figure 3. Effect of cachexia in contralateral gastrocnemius muscle of control and
cachexia-induced rats on the time-course of hydroperoxide-initiated
chemiluminescence. Curves represent means of 6 animals curves. For each animal 80
min curve, a 40 point curve was extracted. Means were compared by a paired
Student’s t test. * p< 0.001, when compared with Control group.
37
Control Day5 Day10 Day14
0,0
0,5
1,0
1,5
2,0
2,5
++
+
**
*
TRAP (μM trolox)
Figure 4. Effect of Walker-256 tumor inoculation on total antioxidant capacity
(TRAP) in homogenate supernatant of rat muscles. Groups were compared by
a paired Student’s t test. Values are expressed as mean ± SE of 6 animals. * p<
0.001, when compared with Control Group;
**
p<0.01, when compared with
Control Group;
+
p<0.001, when compared with Day 5; and
++
p< 0,001 when
com
p
ared with Da
y
10 . Results are ex
p
ressed in TRAP
(μ
M trolóx
)
.
38
Table 2. Levels of glutathione in sham and contralateral gastrocnemius of rats
inoculated with Walker-256 tumor in different times.
Groups GSH GSSG SI
Control 4.040±0.1 0.345±0.04 8.71±2.7
Day 5 1.965±0.2
a
0.310±0. 2 18.98±1.9
b
Day 10 1.490±0.1
a
0.172±0.02
b
11.94±1.0
Day 14 1.267±0.05
a,c
0.378±0.03
c
20.03±2.5
b,c
GSH - Levels of Reduced Glutathione, GSSG - Levels of Oxidized Glutathione, and SI-
Stress Index [(GSSG/GSH-GSSG)x100]. Values represent mean ± SE of 6 animals. p
values, as determined by t-test (two populations) are:
a
p< 0.001, when compared to Control
Group;
b
p<0.05, when compared to Control Group; and
c
p<0.05, when compared to Day
10. Results are expressed in μM / mg protein.
39
Control Day5 Day10 Day14
0,0
0,2
0,4
0,6
0,8
1,0
1,2
USOD. mg protein
-1
*
*
**
#
Figure 5. SOD activity in supernatant from contralateral gastrocnemius
muscles of the control and tumour-bearing rats. These results represents
mean ± SE of 6 animals. p values, as determined by a non-paired t-test (two
populations) are: * p< 0.05, when related with Control Group;
**
p<0.05,
when compared with Day 5; and
#
p<0.05, when compared with Day 10.
40
Control Day5 Day10 Day14
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
*
nmols Carbonyl Protein/
mg total protein
Figure 6. Levels of protein carbonylation on homogenates from
contralateral gastrocnemius muscles of control and tumour-bearing rats.
These results represent means ± SE of 6 animals. p value, as determined by
t-test (two populations) is: * p< 0.05, when compared to Control Group.
41
42
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