Expression of glutathione, glutathione peroxidase and glutathione S-transferase pi in canine mammary tumors

Clinical and molecular characteristics of tumors may assist in the development of more efficient and less toxic therapeutic strategies. In this way, the expression of antioxidant proteins in tumor cells has been investigated as a predictor and prognostic factor in cytotoxic treatment in breast cancer [1].

When there is an excessive production of reactive oxidative species (ROS), the antioxidant defense system is triggered, which is of great importance in the physiopathology of diverse cancer types, including breast cancer [2, 3]. The consequence of this process is the loss of cellular function and progression towards cell death. Moreover, oxidative stress leads to DNA damage and mutations in tumor suppressor genes, events that can be important in the initiation of carcinogenesis [4].

GSH is a tripeptide of glycine, cysteine and glutamic acid that is found in high concentration intracellularly, being the most abundant low molecular mass thiol [5]. Synthesis of GSH requires the consecutive action of two enzymes, first glutamate-cysteine ligase (GCLC) that conjugates glutamic acid and cysteine forming gama-glutamyl cysteine. This compound containing cysteine with a sulfhydryl group (SH) is responsible for the antioxidant activity of GSH. The second reaction is the binding of gama-glutamyl cysteine with glycine by the enzyme glutathione synthetase (GSS), giving rise to the tripeptide gama-glutamyl cysteine glycine-glutathione [6, 7].

GSH-Px is an 80 kDa protein composed of four identical sub-units. Five isoforms of GSH-Px are well characterized in mammals and show tissue-specific distribution. Alteration of these enzyme levels are associated with diverse cancer types, including skin, kidney, intestine and breast cancer [8].

GSTs are a family of intracellular enzymes of detoxification phase II that catalyze diverse electrophilic compounds conjunction to GSH, blocking DNA mutations of the cells [9].

Admittedly, the classification of mammary neoplasms in female dogs is complex and detailed, but it cannot predict the progress of the illness and the survivor time of the animal with cancer nor the chemotherapeutic and/or hormonal treatment feedback. Nowadays, using the possibility of immunohistochemistry diagnosis of the tumors by biomarkers, prognosis can be more accurate and its predictable value can also direct genetic treatment. Besides, the biomarkers that apparently do not have independent prognostic value, together with the clinic-pathologic parameters, may assist in the evaluation of feedback to the patient’s clinical development.

Despite advances in identification of genes involved in tumor growth, progression and resistance to drugs, the way these genes interact with one another remains unclear. Therefore a better understanding of how molecular markers are associated with therapeutic response is indispensable in selecting the correct drugs for treatment. The capacity to establish primary cell cultures of tumors is an important requirement in cancer research, allowing the identification of important prognostic factors and therapeutic agents. Moreover, this is the best way of assessing the predictive value of adjuvant treatment that might also be given to cancer patients [10].

Primary cultures have been successfully used to get answers to drug dosages and to support new anti-cancer drug development [11]. The enzymatic system of glutathione/glutathione transferases (GSH/GSTs) is seen as one of the most important in cellular resistance to multiple drugs (MDR), involving the alteration of GSH level, the expression of genes that codify the enzymes responsible for its synthesis, and the genes that code for GSTs [5].

The aim of this study has been to assess the expression of GSH and the enzymes GSTpi and GSH-Px by immunohistochemistry in mammary tumors and to infer their prognostic value. An in vitro study of molecular analyses was made, verifying the resistance of neoplastic cells to the chemotherapeutic agent.

In this study, there was an increase in GSH expression in tumors without ulceration, not metastatic tumors and low mortality. Our results are in agreement with Buser et al. [12] who demonstrated that high levels of GSH, GSTpi and GSH-Px were associated with favorable clinical characteristics and good prognosis, whereas low levels of GSH and GST activity were associated with more aggressive or more advanced disease.

After 18 months, no significant results were observed when comparing the GSTpi and GSH-Px expression with metastasis and death in our dogs. Our results are in agreement with Jardim et al. [1] where no statistical correlation was found between GSTpi expression and local recurrence and/or metastasis either.

In this study, we verified the influence of chemotherapy with doxorubicin on the GSH and GSH-Px genes expression. According to cell culture growing curves, doxorubicin efficiently causes tumor cell death.

Moreover, doxorubicin treatment reduces the expression of the genes GCLC and GSS, respectively, in treated tumor cells over untreated tumor cells. GCLC sensitizes the enzyme glutamate cysteine ligase to catalyze the first step for GSH synthesis and GSS codes for glutathione synthetase catalyzing the second and last step of synthesis of GSH. Together, these data support the notion that doxorubicin reduces a pattern of gene expression associated with modulation of the antioxidant capacity of mammary tumor cells.

Many studies have shown alterations in GSH activity after in vitro treatment with doxorubicin or similar drugs. Our results agree with Ozkan and Fiskin [13], in which epirubicin - a structural analog of doxorubicin - decreases GSH activity after 24 h in cultured mammary neoplastic cells.

Some studies suggest that reactive oxygen species (ROS) production in doxorubicin treatment is responsible for cytotoxicity in neoplastic cells, which could induce the synthesis of antioxidant enzymes, such as GSH and GSH-Px, making the neoplastic cells resistant to oxidative damage [14]. In the same way, Han et al. [15] found greater sensitivity to doxorubicin when the GSH levels decreased in the mammary carcinoma cell line (MCF-7). However, in contrast, Di et al. [16] demonstrated that GSH overexpression did not prevent tumor cell apoptosis after treatment with doxorubicin, suggesting that the cytotoxicity of the drug might not be wholly related to ROS production.

Additionally, the involvement of GSH in the therapeutic response to doxorubicin is controversial. Some authors suggest that GSH inhibition leads to the drug accumulating in the nucleus of the tumor cells [17, 18], whereas others suggest that resistance to doxorubicin and other similar drugs is not directly related to the binding of the drug to GSH [14].

Exactly what intracellular alterations are responsible for the MDR phenotype in neoplastic cells remain largely unknown. There is a suggestion that high concentrations of GSH, GSH-Px and GSTpi, independent of other intracellular alterations, do not decisively contribute to MDR [19, 20].

The resistance to the antineoplastic agent cisplatin, for instance, has been associated to the high expression of the enzyme gama glutamyl transpeptidase (GGT) localized at the cell membrane, which is responsible for its degradation. Thereby, despite the fact that cytotoxic treatment decreases intracellular GSH concentration, this enzyme hydrolyzes serum GSH, releasing the necessary amino acids for intracellular GSH replenishment [21]. The enzyme GGT is mainly found in liver and kidney cells; however, resistance to drugs due to high levels of this enzyme has been found in many cancer types including some mammary tumors [21].

In this context, there are many studies trying to identify agents that can increase the efficiency of conventional drugs and/or reverse the MDR phenotype in neoplastic cells [22, 23]. Despite the correlation between high expression of these genes and the MDR, some studies suggest that using agents that increase GSH, GSH-Px and GSTs levels during drug treatment is a promising way to reduce oxidative stress, thus reducing the collateral effects of chemotherapy in breast cancer patients [24–26].

The mammary tumors cultivated in vitro showed underexpression of the GSH coding genes and no alteration to the expression of GSH-Px, after treatment with doxorubicin. This can be explained by the interaction between these two enzymes in the antioxidant defense of tumor cells. GSH-Px has selenium at its catalytic site and uses GSH as an electron donor for the reduction of H₂O₂ to H₂O [12, 27]. So the results show that GSH and GSH-Px don’t provide adequate defense against oxidants, rendering them sensitive to drugs.

In this context, it is necessary to clarify the mechanism of these enzymes in cell resistance and their true prognostic value [28, 29]. Knowledge of GSH expression and its enzymes in a wide range of biology processes is expanding. Advances in analytical techniques and protein localization will present many opportunities for the development of therapeutic interventions in cancer and other diseases related to levels of oxidative stress [5].

High expression of GSH in neoplastic mammary cells from female dogs is associated with the best clinical outcome, including a greater survival time. Therefore this high expression plays an important role in the clinical outcome of patients with mammary tumors and can be used as prognostic marker for mammary neoplasms in female dogs.

Treatment of mammary neoplastic cells with doxorubicin in vitro is an efficient way to induce cell death, reducing the expression of the GCLC and GSS genes that are related to antioxidant production. Thus they can be considered potential predictive markers on the therapeutic feedback to breast cancer. More studies, using large cohorts in future studies, are needed to confirm the prognostic value of these markers in the mammary tumor progression in female dogs.