Evaluation of trace elements associated with antioxidant enzymes in blood of primary epithelial ovarian cancer patients

Highlights

• Trace elements may have a critical role in ovarian cancer development through AOEs.

• Mn and Se levels were decreased in all histologic subtypes of EOC.

• Only Mn levels were changed according to tumor grading in serous EOC.

• The correlation between Cu and Se levels was remarkable in G 1-2 tumors of M1.

• High erythrocyte Cu/Se ratio might be a favourable marker for EOC.

Abstract

Epithelial ovarian cancer (EOC) has been associated with oxidative stress (OS) due to epithelial inflammation which makes ovaries more vulnerable to the deleterious effects of reactive oxygen species (ROS). However, antioxidant enzymes (AOEs) such as manganese-superoxide dismutase (Mn-SOD), copper,zinc-superoxide dismutase (Cu,Zn-SOD) and glutathione peroxidase (GPx1) protect cells against the biological damage of ROS-induced OS and support cancer prevention by maintaining normal cell cycle progression, inhibiting proliferation, tumor invasion, angiogenesis, inflammation or inducing apoptosis.

In the present study, we aimed to measure the levels of trace elements [manganese (Mn), copper (Cu), zinc (Zn) and selenium (Se)] which are structurally and/or functionally associated with the AOEs by inductively coupled plasma/mass-spectrometry (ICP/MS) in blood samples of patients with EOC (M, n = 26) and compare the data with healthy subjects (C, n = 46). Serous EOC (M1, n = 18) data were also evaluated according to the tumor grading [well or moderately well differentiated (G 1-2) vs. poorly differentiated or undifferentiated (G3)] and staging of disease [stage I-II (SI-II) vs. stage III (SIII)]. We obtained; i) The Mn and Se levels of M were significantly lower than C, ii) only Mn levels were changed [(G3_(Mn) < G 1-2 _(Mn)] in M1, iii) significant correlations were observed between [Cu and Zn levels (r = 0.701, p = 0.036) in G 1-2 and (r = 0.686, p = 0.041) in G3; Cu and Se levels (r = 0.960, p = 0.000) in G3; Mn levels and Mn-SOD expression (r = 0.551, p = 0.006) in M, (r = 0.857, p = 0.007) in G 1-2 and (r = 0.690, p = 0.056) in G3; Se levels and erythrocyte GPx1 activity (r = 0.660, p = 0.053) in G 1-2 ; Se levels and erythrocyte Cu,Zn-SOD activity (r = 0.693, p = 0.038) in G3]. The study revealed that trace elements, particularly low Mn and Se levels along with high Cu/Se ratios might be of value in all histologic subtypes of EOC. Although Mn level was important in terms of discriminating tumor grades, positive correlation between Cu-Se levels was also remarkable in patients with G 1-2 tumors of M1. Moreover, high erythrocyte Cu/Se ratios might be a favourable marker for EOC.

Introduction

Carcinogenesis is a multifactorial diease due to genetic and epigenetic alterations facilitated by various risk factors including environmental factors such as toxic heavy metals [arsenic (As), cadmium (Cd), lead (Pb), mercury (Hg), nickel (Ni), etc.] and trace elements [manganese (Mn), copper (Cu), zinc (Zn), selenium (Se), iron (Fe), etc]. The carcinogenicity of metals and their compounds/reactive metabolites mostly depends on their bioavailability in biological systems and their interference with (i) cellular redox regulation and antioxidant defense systems resulting in generally reactive oxygen species (ROS)-induced oxidative stress (OS), enhanced lipid peroxidation (LPO), changes in calcium and sulphydril homeostasis (ii) DNA repair mechanisms resulting in genomic instability/accumulation of important mutations, (iii) elements of signal transduction pathways such as protein kinases, transcription factors, etc. resulting in deregulation of cell proliferation, (iv) gene expression by activating proto-oncogenes or inactivating cell growth controls such as tumor supressor genes resulting in stimulated cell proliferation, (v) autophagy and related proteins resulting in dysfunction and cellular transformation or inflammation [[1], [2], [3], [4], [5], [6]].

Numerous in vivo and in vitro studies have pointed out to adverse effects of metals on female reproductive system and scientific consensus reached that high metal exposure/deficiency is associated with some reproductive disorders/diseases [[7], [8], [9], [10], [11], [12], [13], [14]] such as infertility, spontaneous abortions, preterm deliveries, uterine fibroids [15], endometriosis [16], polycystic ovarian syndrome [17] or gynecological cancers [15,[18], [19], [20], [21], [22]]. Ovarian cancer is the leading cause of death among gynecological cancers. Thus, it is a life-threatining disease with respect to its histopathology, clinical characteristics and high incidence/mortality rates increased with age. Epithelial ovarian cancer (EOC) comprises 90% of malignant and 60% of all ovarian tumors and 75% of EOC are characterized by serous histologic subtype [[23], [24], [25], [26]].

Epithelial ovarian tumors are associated with reactive oxygen species (ROS) induced-oxidative stress (OS) due to epithelial inflammation of the ovaries arising from incessant ovulation [27,28]. Excessive ROS have a paradoxical role in tumorigenesis with their deleterious effects to the body such as oxidative damage to biomolecules, genomic instability, uncontrolled growth, and perturbed differentiation [4,[29], [30], [31]] and anti-tumorigenic effects against tumor cells such as induction of cellular senescence and apoptosis [4,[32], [33], [34]]. ROS are involved in all stages of cancer development; initiation, promotion and progression. A disruption in the redox homeostasis due to high ROS levels and depletion or decreased removing capacity of endogeneous antioxidants might lead to cancer [28,30,31,35]. There are various processes in the body to alleviate ROS and counteract detrimental effects of ROS induced-OS on important biomolecules. These mechanisms include antioxidant enzymes (AOEs) which protect aerobic cells against biological damage of ROS and support cancer prevention by modulating signal transduction pathways [4,[29], [30], [31],35,36].

Some trace elements such as Mn, Cu, Zn, Se and Fe are essential for normal formation or functions of the body and exist in the structure of AOEs. Thus, they play critical roles in important biological processes, but also contribute to the development of many chronic diseases, including cancer with their excessive levels (e.g. Cu, Fe, Se) or loss of antioxidant feature depending on deficiency (e.g. Zn, Se) [2,[16], [17], [18], [19],37].

The main AOEs associated with trace elements are superoxide dismutases (SODs) in the body. They dismutate superoxide anion into hydrogen peroxide (H₂O₂) by requiring catalytic cycle of redox active transition metals (i.e. oxidized Mn⁺³/reduced Mn⁺²) in their active sites. Manganese-superoxide dismutase (Mn-SOD) has one Mn⁺² ion in its each monomeric subunit, whereas copper,zinc-superoxide dismutase (Cu,Zn-SOD) has two subunits each containing one Cu⁺² and one Zn⁺² ions. The Cu⁺² is a redox active metal and responsible for the catalytic activity of Cu,Zn-SOD with its catalytic cycle (Cu⁺³/Cu⁺²), while Zn⁺² enhances the stability of this enzyme and provides a wide range of pH value for its activity [[38], [39], [40], [41], [42]]. Cu can form complexes with gonadotropin releasing hormones and high levels of FSH and LH are associated with ovarian tumor development [7]. Another group of AOEs is glutathione peroxidase (GPx1) which is Se-dependent and carries 4 atom gram of Se in each molecule in the form of selenocysteine. Organic hydroperoxides, including H₂O₂ are converted and detoxified by these seleno-enzymes mainly through oxidation of reduced glutathione (GSH) to oxidized glutathione (GSSG) [[43], [44], [45]]. Alterations in the GSH:GSSG ratio may modulate Cu homeostasis in the body due to the critical roles of GSH in cellular redox balance, Cu binding and Cu delivery or flux [40]. Besides this, Se and selenoproteins like GPx1 are elevated in large healthy follicles and pronounced to have a critical role as an antioxidant during late follicular development [46]. Zn has also antioxidant properties as a cofactor of important enzymes, including Cu,Zn-SOD. It modulates total cellular GSH concentration by altering the expression of glutamate-cysteine ligase, inhibits the pro-oxidant enzyme NADPH-oxidase and induces the synthesis of toxic heavy metal binding protein metallothionein [47,48]. Zn also functions as an anti-inflammatory agent by reducing inflammatory cytokine production [48]. The studies pointed out that both Se and Zn deficiencies are in association with disturbed female reproductive system functions such as unexplained infertility, gestational complications, miscarriages, low oocyte maturation or anovulation [49,50].

Therefore, in the present study, firstly we aimed to measure trace element levels (Mn, Cu, Zn, Se) which are structurally and/or functionally associated with the AOEs in blood samples of patients with EOC and then evaluated the possible correlations between these trace elements and their relevant AOEs together with OS-induced LPO markers. This is the first study evaluating trace elements in patients with EOC according to histopathological and clinical characteristics of the disease by identifying also the relationship between these elements and relevant AOEs requiring them for enzymatic activity or stability.