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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2012 Feb 24;138(6):999–1009. doi: 10.1007/s00432-012-1176-4

Apoptosis, angiogenesis, inflammation, and oxidative stress: basic interactions in patients with early and metastatic breast cancer

Enas A Hamed 1,✉, Madeha M Zakhary 2, Doaa W Maximous 3
PMCID: PMC11824265  PMID: 22362301

Abstract

Purpose

Breast cancer (BC) is a complex, multi-stage disease involving deregulation of different signaling cascades. The present study was conducted to determine the extent of apoptosis, angiogenesis, inflammation, and oxidative stress in patients with different stages of BC as an approach to disease biological behavior. Therefore, plasma levels of soluble (s) Fas, bcl-2 as antiapoptotic indices; interleukin (IL)-8, tumor necrosis factor (TNF)-α as apoptotic, inflammatory, angiogenic indices; lipid peroxides (LPO), nitric oxide (NO) as oxidative stress and angiogenic indices were measured in patients with BC.

Methods

Thirty-seven newly diagnosed patients with BC, 30 patients with benign breast masses, and 30 healthy controls were recruited. Plasma levels of sFas, bcl-2, IL-8, and TNF-α were measured by immunosorbent assay kits and LPO and NO by chemical methods.

Results

Plasma sFas and LPO were significantly higher in BC patients versus benign breast masses and healthy controls (P < 0.0001). Bcl-2, IL-8, TNF-α, and NO were significantly higher in benign breast masses (P < 0.0001, P < 0.037, P < 0.0001, P < 0.001) and BC (P < 0.0001) versus controls and in BC versus benign breast masses (P < 0.0001). sFas, bcl-2, IL-8, TNF-α, LPO, and NO were increased with advanced tumor stages. There were positive correlations between sFas, bcl-2, IL-8 TNF-α, LPO, and NO.

Conclusions

BC tumor cells overexpress bcl-2 and sFas to secure their outgrowth and survival. However, this coincides with activation of physiologic regulatory mechanisms, as increased IL-8, TNF-α, LPO, and NO, which try to stop tumor cells by inducing apoptosis. Outcompeting of these mechanisms result in tumor progression as IL-8, TNF-α, and NO are also angiogenic stimulators.

Keywords: Angiogenesis, Apoptosis, Breast cancer, Cytokines, Oxidative stress

Introduction

Breast cancer (BC) is a complex, multi-stage disease involving the deregulation of a number of different signaling cascades. Although significant improvements in BC therapy have occurred recently, most deaths from BC are still caused by metastases that are resistant to conventional treatment. Therefore, demands for therapy to increase efficiency of radiation and chemotherapy are needed. Apoptosis is known to participate in various biological processes such as development and maintenance of tissue homeostasis and elimination of cancer cells. Recent studies have implicated apoptosis as a common mechanism through which chemotherapeutic agents exert their cytotoxicity (Thomadaki and Scorilas 2008). Fas (Apo-1, CD95) is a cell-surface receptor that is a member of the tumor necrosis factor (TNF)/nerve growth factor receptor superfamily and mediate apoptosis through binding of FAS ligand (FASL) or agonistic anti-FAS antibody. Fas is found in two forms, transmembrane and soluble. The soluble form of Fas (sFas) inhibits Fas-mediated apoptosis by neutralizing FasL or anti-Fas antibody. Increased concentration of serum sFas has been reported in various diseases, including neoplastic diseases. These findings suggest that cancer cells upregulate or stimulate sFas production to protect themselves from Fas-mediated apoptosis (Natoli et al. 1995). In addition to Fas, several other proteins play a major role in regulating apoptosis. For example, the bcl-2 gene family contains many related and interacting molecules with antiapoptotic (e.g., bcl-2) and pro-apoptotic (e.g., Bax, Bid, and Bak) activities. Bcl-2, an antiapoptotic survival factor, is a major protein protecting the integrity of mitochondria, and its expression has been detected in 40–80% of primary breast cancers (Nahta and Esteva 2003). Its tumorigenic potential has been demonstrated in animal models (McDonnell and Korsmeyer 1991) and is supported by the finding of over-expression of bcl-2 in a variety of tumors and in lymphomas in which bcl-2 acts as an oncogene (Pietenpol et al. 1994). Targeting bcl-2 in tumor cells could enhance the effects of chemotherapy.

Oxidative stress and inflammation are closely associated with tumor growth. Cytokines stimulate cancer cell growth and contribute to locoregional relapse and metastases (Kozlowski et al. 2003). Interleukin (IL)-8, a member of the CXC chemokine family, initially shown to be a chemoattractant for neutrophils and lymphocytes, can act multifunctionally to induce tumor growth and metastasis. Further, IL-8 secreted by tumor cells may also promote vascularization by enhancing endothelial cell proliferation, survival, and matrix metalloproteinases (MMPs) production. Tumor biological experiments suggest that IL-8 is involved in breast cancer cell invasion and angiogenesis (Lin et al. 2004). TNF-α is a peptide hormone that affects tumor cell necrosis, inflammation, and immune response (Gong et al. 2005). It is worth to note that TNF-α acts as a mediator of the apoptotic process and has selective cytotoxicity against malignant breast tumor cells, promoting an apoptotic type of cell death in MCF-7 cells (Donato and Klostergaard 2004). TNF-α has been postulated as a key player in the tumor microenvironment but has a paradoxical role in disease evolution: It can act both as a necrotic or as a promoting factor, for example, the endogenous TNF-α chronically produced in the tumor microenvironment enhances both tumor development and spreading, while local administration of high-doses of TNF-α is antiangiogenic and has a powerful anti-tumoral effect (Balkwill 2002).

Oxidative stress caused by increased free radical generation and/or decreased antioxidant level in the target cells and tissues has been suggested to play an important role in carcinogenesis (Haklar et al. 2001). Prime targets of free radicals are the polyunsaturated fatty acids in cell membranes, and their interaction results in lipid peroxidation. Changes in the concentration of lipid peroxides (LPO) have been reported in breast cancer (Khanzode et al. 2004). Nitric oxide (NO) is produced from l-arginine by the family of nitric oxide synthase (NOS) enzymes, forming the free radical NO and citrulline as a byproduct. Three distinct isoforms of the NOS enzyme have been isolated and represent the products of three different genes. Two of these are constitutively expressed in a variety of cells and are calcium dependent. A third, an inducible (iNOS), calcium independent form, is important in the immunogenic and cytotoxic response of T-lymphocytes and macrophages (Geller and Billiar 1998). Nitric oxide is involved in tumor cell apoptosis and has additional effects on tumor blood flow, immune responses, and angiogenesis (Mortensen et al. 1999). Analysis of nitrite and nitrate has been used extensively as an index of endogenous production of NO in biological systems, with respect to various pathological processes (Thomsen et al. 1995).

The present cross-sectional study was conducted to determine the extent of apoptosis versus angiogenesis together with inflammatory mediators and degree of oxidative stress in breast cancer patients. This was done by determination of sFas and bcl-2 as antiapoptotic indices as well as IL-8 and TNF-α (angiogenic, inflammatory, and apoptotic index), NO (oxidative stress, angiogenic, and apoptotic index), and LPO (oxidative stress index) in plasma of patients with malignant as well as those with benign breast masses. The levels of these indices would be correlated with host and tumor characteristics.

Materials and methods

Patients

Thirty-seven female newly diagnosed BC patients and 30 female patients with benign breast masses, confirmed by pathological examination, were recruited in this cross-sectional study from surgical clinic, South Egypt Cancer Institute, Assiut, Egypt, during January to December 2010. Patients were recruited before surgery, radiotherapy, and chemotherapy. The breast cancer patients had age between 30 and 60 years [mean ± standard deviation (SD), 44.73 ± 9.42 years]; those with the benign breast masses were aged between 25 and 60 years (mean ± SD, 45.60 ± 10.52 years). The healthy control subjects were completely healthy and were selected from screening clinics consisting of 30 individuals with age between 35 and 60 years (mean ± SD, 47.40 ± 8.24 years). All participants were subjected to thorough evaluation in the form of complete history taking, physical examination, complete blood count and serum chemistry analysis, chest X-ray, abdominal ultrasound, and/or computed tomography when indicated. The data as age, menopausal state, breast appearance, site of breast masses, estrogen receptor (ER) status, human epidermal growth factor receptor-2 (Her-2) status, maximum tumor size, fixation of the mass to chest wall, and tumor, nodes, and metastases (TNM) staging were collected. According to TNM staging, there were 6 cases (16.22%), 14 (37.84%), 9 (24.32%), and 8 (21.62%) breast cancer patients classified as stages I, II, III, and IV, respectively. Histopathological examination showed that 35 (94.60%) patients had invasive ductal carcinoma and 2 (5.40%) had medullary carcinoma. Of the 30 patients with benign breast diseases, 8 (26.67%) had duct ectasia, 18 (60.00%) had fibrocystic disease, and 4 (13.33%) had chronic breast abscess. None of the patients were using oral contraceptives, hormones, vitamins, steroids, or anti-inflammatory medications, and all were nonsmokers. None of them had concomitant diseases such as diabetes mellitus, liver diseases, and rheumatoid arthritis. The selection criteria of the controls included no history of cancer or benign breast diseases or family history of breast cancer or autoimmune disorders and matching to patient’s age. In accordance with the declaration of Helsinki, the study protocol was approved by the local ethical committee and written informed consent was obtained from all participants. The regiment of treatment was as follows: Patients with stages I and II underwent modified radical mastectomy (MRM) and then received adjuvant chemotherapy (6 cycles) and chemotherapy, while patients with stage III received neoadjuvant chemotherapeutic agents (3 cycles) for down-staging followed by MRM and another 3 cycles of chemotherapy as adjuvant therapy conjoined with radiotherapy.

Samples and assay

Peripheral venous blood samples (6 ml) were collected by venipuncture before any therapeutic intervention on the day of surgery, using ethylenediaminetetraacetic acid as anticoagulant, and the plasma was separated by centrifuging at 3,000 rpm for 10 min and stored at −20°C until use. Plasma sFas levels were measured by use of sFas ELISA kit (Code No. 5251, Medical and Biological Laboratories Co Ltd, Tokyo, Japan) with sensitivity 0.5 ng/ml. The average within-run and between-run coefficients of variation (CVs) were 4.5 and 5.2%, respectively. Bcl-2 was determined by ELISA kit supplied by Calbiochem (Cat No. QIA23, Darmstadt, Germany), with sensitivity <1 U/ml. IL-8 level was determined by ELISA kit (Code No. 237, Immunotech SAS Company, Marseille Cedex, France) with sensitivity 8 pg/ml; intra-assay and inter-assay coefficients of variation ranged between 2.3 and 5.5% and between 7.6 and 10.1%, respectively. TNF-α was determined by ELISA kit (Cat No. SK00109-01, Beijing Adipobiotech Santa Clara, CA, USA) with sensitivity 4 pg/ml; intra-assay and inter-assay precision were 4 and 8%, respectively. The levels of LPO were measured as thiobarbituric acids reactivity. The product of the reaction between malondialdehyde and thiobarbituric acid was measured as described by Grau et al. (2000) The levels of NO was determined as total nitrite after deproteinization with ZnSO4 (30%), and color developed by reaction with Griess reagent (1% sulfanilamide/0.1% naphthyelthylene diamine diHCL, w/v in 2.5% H3PO4) was recorded at 550 nm against reagent blank using sodium nitrite 10–100 μM as standard (Ding et al. 1988). Estrogen receptor and Her-2 status were determined by immunohistochemical staining method.

Statistical analysis

Statistical Science for Social Package (SPSS Inc, USA) software computer program version 16 was used for data analysis. Data were presented as mean ± standard deviation or number and percentage (n, %) as appropriate. For comparison of two groups, the nonparametric test for independent variables was used while comparisons of multiple groups were done using one-way analysis of variation and Kruskal–Wallis tests for parametric and nonparametric variables, respectively. Spearman’s and Pearson’s correlation tests were used as appropriate for correlating nonparametric and parametric variables. For all tests, a probability P < 0.05 was considered significant.

Results

Table 1 showed the demographic and clinical characteristics of all studied groups. There was no significant difference regarding age between the studied groups. Postmenopausal state was found in 50.00% (n = 15), 33.33% (n = 10), and 54.10% (n = 20) of healthy controls, benign breast masses patients, and BC patients, respectively. In BC, mass fixed to the breast in 29.73% (n = 11), mass fixed to chest wall in 37.80% (n = 14), positive Her-2 in 59.50% (n = 22), and positive ER in 54.10% (n = 20).

Table 1.

Demographic and clinical characteristics of all participants

Variables Healthy controls (n = 30) Benign breast masses (n = 30) Malignant breast tumors (n = 37)
Age (years) 47.40 ± 8.24 45.60 ± 10.52 44.73 ± 9.42
35.00–60.00 25.00–60.00 30.00–60.00
*P < 0.462

*P < 0.252

**P < 0.708
Postmenopausal state 15 (50.00%) 10 (33.33%) 20 (54.10%)
 Side
 Right – 12 (40.00%) 17 (45.90%)
 Left – 18 (60.00%)

20 (54.10%)

**P < 0.272
Size of breast mass (cm2) 24.33 ± 22.38 35.50 ± 34.11
6.00–100.00 4.00–121.00
**P < 0.128
Positive Her-2 – – 22 (59.50%)
Positive estrogen receptor – – 20 (54.10%)
Fixed to breast – – 11 (29.73%)
Fixed to chest wall – – 14 (37.80%)
TNM tumor stage
 I – – 6 (16.22%)
 II – – 14 (37.84%)
 III – – 9 (24.32%)
 IV – – 8 (21.62%)
Pathology of masses
 Invasive ductal carcinoma – – 35 (94.60%)
 Medullary carcinoma – – 2 (5.40%)
 Duct ectasia – 8 (26.67%) –
 Fibrocystic disease – 18 (60.00%) –
 Chronic breast abscess – 4 (13.33%) –
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Data are expressed as mean ± SD or number (%) as appropriate

Her-2 human epidermal growth factor receptor-2, TNM tumor, nodes, and metastases

* P < significance versus control, ** P < significance versus benign tumor

Plasma sFas and LPO were significantly higher in patients with BC versus those with benign breast masses and healthy controls (P < 0.0001 for all). Bcl-2, IL-8, TNF-α, and NO were significantly higher in benign breast masses (P < 0.0001, P < 0.037, P < 0.0001, P < 0.001) and BC (P < 0.0001 for all) versus controls and in malignant versus benign breast masses (P < 0.0001 for all) (Table 2).

Table 2.

Comparison of measured apoptotic, inflammatory, and oxidative stress markers between different studied groups

Variables Healthy controls (n = 30) Benign breast masses (n = 30) Malignant breast tumors (n = 37)
Soluble Fas (ng/ml) 1.27 ± 0.36 2.27 ± 0.41 6.50 ± 3.48
0.70–2.10 1.70–3.00 2.00–12.10
*P < 0.079 *P < 0.0001
**P < 0.0001
Bcl-2 (U/ml) 76.02 ± 10.80 112.17 ± 22.85 181.44 ± 43.89
50.40–90.80 77.50–137.90 105.70–263.80
*P < 0.0001

*P < 0.0001

**P < 0.0001
IL-8 (ng/ml) 80.29 ± 10.91 97.06 ± 9.63 165.28 ± 47.90
60.80–101.20 81.30–115.60 102.40–259.40
*P < 0.037

*P < 0.0001

**P < 0.0001
TNF-α (pg/ml) 68.55 ± 18.21 132.21 ± 21.75 218.02 ± 52.93
28.60–92.10 98.50–163.10 132.30–316.70
*P < 0.0001

*P < 0.0001

**P < 0.0001
Lipid peroxide (μmol/l) 2.36 ± 0.55 3.47 ± 0.45 8.19 ± 4.19
1.40–3.20 2.80–4.20 3.10–16.70
*P < 0.105

*P < 0.0001

**P < 0.0001
Nitric oxide (μmol/l) 4.32 ± 0.92 6.12 ± 1.40 14.29 ± 2.77
1.50–6.30 4.70–9.60 10.30–20.80
*P < 0.001

*P < 0.0001

**P < 0.0001
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Data are expressed as mean ± SD

IL-8 interleukin-8, TNF-α tumor necrosis factor-alpha

* P < significance versus control, ** P < significance versus benign tumor

In patients with BC, patients with advanced disease as evidence by fixed masses within the breast and those with fixed masses to the chest wall showed significant elevated plasma levels of sFas, bcl-2, IL-8, TNF-α, LPO, and NO compared to those with mobile masses to the breast (P < 0.0001, P < 0.005, P < 0.0001, P < 0.001, P < 0.0001, P < 0.007) and those with not fixed masses to chest wall (P < 0.0001, P < 0.003, P < 0.0001, P < 0.0001, P < 0.0001, P < 0.002), respectively (Table 3).

Table 3.

Comparison of measured apoptotic, inflammatory, and oxidative stress markers between different subgroups of malignant breast cancer patients

Variables Menopausal state Mobility within breast Relation to chest wall Her-2 Estrogen receptor
Pre (n = 20) Post (n = 17) Mobile (n = 26) Fixed (n = 11) Mobile (n = 23) Fixed (n = 14) Positive (n = 22) Negative (n = 15) Positive (n = 20) Negative (n = 17)
Soluble Fas (ng/ml) 5.96 ± 3.35 7.13 ± 3.64 5.27 ± 3.05 9.41 ± 2.68 4.93 ± 2.81 9.08 ± 2.95 6.05 ± 3.48 7.16 ± 3.50 7.23 ± 3.61 5.64 ± 3.23
P < 0.318 P < 0.0001 P < 0.0001 P < 0.349 P < 0.170
Bcl-2 (U/ml) 171.89 ± 38.19 192.68 ± 48.52 168.72 ± 39.52 211.50 ± 40.21 165.32 ± 38.56 207.91 ± 40.04 174.07 ± 43.90 192.25 ± 43.04 188.38 ± 46.76 173.27 ± 40.08
P < 0.154 P < 0.005 P < 0.003 P < 0.221 P < 0.303
IL-8 (ng/ml) 155.52 ± 44.92 176.76 ± 50.06 146.50 ± 37.23 209.66 ± 41.35 141.70 ± 36.58 204.01 ± 38.60 166.24 ± 51.60 163.87 ± 43.61 179.01 ± 51.33 149.12 ± 38.99
P < 0.182 P < 0.0001 P < 0.0001 P < 0.885 P < 0.057
TNF-α (pg/ml) 208.74 ± 47.52 228.94 ± 58.20 200.17 ± 46.74 260.20 ± 42.85 194.21 ± 45.57 257.13 ± 39.91 215.98 ± 55.80 221.01 ± 50.17 231.37 ± 58.63 202.32 ± 41.67
P < 0.253 P < 0.001 P < 0.0001 P < 0.781 P < 0.097
Lipid peroxide (μmol/l) 7.52 ± 4.04 8.98 ± 4.35 6.67 ± 3.43 11.78 ± 3.71 6.32 ± 3.24 11.26 ± 3.82 7.80 ± 4.46 8.75 ± 3.85 9.15 ± 4.38 7.06 ± 3.79
P < 0.297 P < 0.0001 P < 0.0001 P < 0.505 P < 0.133
Nitric oxide (μmol/l) 13.97 ± 2.62 14.68 ± 2.96 13.53 ± 2.36 16.11 ± 2.91 13.23 ± 2.34 16.04 ± 2.57 14.22 ± 2.89 14.41 ± 2.67 14.88 ± 3.07 13.61 ± 2.25
P < 0.439 P < 0.007 P < 0.002 P < 0.842 P < 0.166
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Data are expressed as mean ± SD

P: significance versus subgroup. IL-8 interleukin-8, TNF-α tumor necrosis factor-alpha, Her-2 human epidermal growth factor receptor-2

The plasma levels of sFas, bcl-2, IL-8, TNF-α, LPO, and NO were significant higher in patients with malignant breast cancer stages III and IV versus those with stages I and II and also in patients with stage IV versus patients with stage III. Meanwhile, plasma levels of bcl-2, TNF-α, and NO were significantly higher in patients with stage II versus patients with stage I (P < 0.035, P < 0.018, P < 0.046) (Table 4).

Table 4.

Comparison of measured apoptotic, inflammatory, and oxidative stress markers between subgroups of malignant breast cancer according to tumor, nodes, and metastases (TNM) stages

Variables Tumor stage
Stage I (n = 6, 16.22%) Stage II (n = 14, 37.84%) Stage III (n = 9, 24.32%) Stage IV (n = 8, 21.62%)
soluble Fas (ng/ml) 2.90 ± 0.76 4.14 ± 1.22 8.16 ± 2.13 11.45 ± 0.57
*P < 0.069

*P < 0.0001

**P < 0.0001

*P < 0.0001

**P < 0.0001

***P < 0.0001
Bcl-2 (U/ml) 132.15 ± 19.10 158.39 ± 16.17 201.34 ± 25.93 236.35 ± 35.94
*P < 0.035

*P < 0.0001

**P < 0.0001

*P < 0.0001

**P < 0.0001

***P < 0.006
IL-8 (ng/ml) 122.12 ± 20.20 135.18 ± 25.62 182.22 ± 28.12 231.25 ± 25.16
*P < 0.300

*P < 0.0001

**P < 0.0001

*P < 0.0001

**P < 0.0001

***P < 0.0001
TNF-α (pg/ml) 158.18 ± 16.75 190.51 ± 28.42 236.06 ± 33.28 290.75 ± 19.83
*P < 0.018

*P < 0.0001

**P < 0.0001

*P < 0.0001

**P < 0.0001

***P < 0.0001
Lipid peroxide (μmol/l) 3.97 ± 0.51 5.39 ± 1.39 9.90 ± 2.47 14.31 ± 1.46
*P < 0.087

*P < 0.0001

**P < 0.0001

*P < 0.0001

**P < 0.0001

***P < 0.0001
Nitric oxide (μmol/l) 11.40 ± 0.94 13.11 ± 1.77 14.79 ± 1.72 17.99 ± 1.90
*P < 0.046

*P < 0.001

**P < 0.026

*P < 0.0001

**P < 0.0001

***P < 0.0001
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Data are expressed as mean ± SD

IL-8 interleukin-8, TNF-α tumor necrosis factor-alpha

* P significance versus stage I, ** P significance versus stage II, *** P significance versus stage III

There were significant positive correlations between sFas, bcl-2, IL-8, TNF-α, LPO, NO, fixation of the mass within breast, fixation of the mass to chest wall, and advanced tumor stages. Significant negative correlations were found between positive ER and age, menstrual state, and fixation to breast (r = −0.802, P < 0.0001, r = −0.850, P < 0.0001, r = −0.362, P < 0.028) (Table 5).

Table 5.

Correlations between demographic, clinical characteristics and measured apoptotic, inflammatory and oxidative stress markers in malignant breast cancer patients

Variables sFas Bcl-2 IL-8 TNF-α Lipid peroxide NO Age Menstrual state Her-2 Estrogen receptors Fixation to breast Fixation to chest wall
Bcl-2 0.918
0.0001
IL-8 0.892 0.769
0.0001 0.0001
TNF-α 0.899 0.818 0.940
0.0001 0.0001 0.0001
Lipid peroxide 0.985 0.899 0.909 0.903
0.0001 0.0001 0.0001 0.0001
NO 0.831 0.732 0.857 0.893 0.855
0.0001 0.0001 0.0001 0.0001 0.0001
Age 0.245 0.311 0.238 0.223 0.259 0.246
0.144 0.061 0.156 0.184 0.122 0.142
Menstrual state 0.169 0.239 0.224 0.193 0.176 0.131 0.871
0.318 0.154 0.182 0.253 0.297 0.439 0.0001
Positive Her-2 0.158 0.206 −0.025 0.047 0.113 0.034 0.112 0.012
0.349 0.221 0.885 0.781 0.505 0.842 0.510 0.944
Positive estrogen receptors −0.230 −0.174 −0.315 −0.277 −0.251 −0.233 −0.802 −0.850 0.012
0.170 0.303 0.057 0.097 0.133 0.166 0.0001 0.0001 0.944
Fixation to breast 0.551 0.452 0.611 0.526 0.565 0.433 0.321 0.231 −0.055 −0.362
0.000 0.005 0.000 0.001 0.000 0.007 0.052 0.169 0.745 0.028
Fixed to chest wall 0.586 0.477 0.640 0.584 0.579 0.500 0.200 0.063 −0.077 −0.160 0.834
0.0001 0.003 0.0001 0.0001 0.0001 0.002 0.235 0.709 0.652 0.344 0.0001
Advanced tumor staging 0.907 0.842 0.836 0.869 0.904 0.799 0.314 0.180 0.118 −0.260 0.676 0.719
0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.058 0.286 0.485 0.120 0.0001 0.0001
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IL-8 interleukin-8, TNF-α tumor necrosis factor-alpha, NO nitric oxide, Her-2 human epidermal growth factor receptor-2

Discussion

Apoptosis is a fundamental mechanism that regulates cell and tissue homeostasis and eliminates cancer cells. It is generally believed that insufficient apoptosis causes cancer. Extrinsic and intrinsic-mediated pathways lead to apoptosis. The extrinsic-mediated pathway is also called the receptor-mediated pathway. It is characterized by the activation of cell surface ligand-gated death receptors, including those of the superfamily of TNF receptors, such as TNFR, Fas, and TRAIL receptors. When specific ligands bind to the extracellular domain of death receptors, receptor trimerization will be triggered. The TRADD and the FADD adaptor molecules then bind to the cytoplasmic domain of the receptor. Initiator caspase-8 then causes activation and further cleaves and activates effector caspase-3, inducing irreversible cell death (Thorburn 2004). The intrinsic or mitochondria mediated pathway can be activated directly without being triggered by a death receptor. In this process, mitochondria will be disrupted by cell stress, releasing of cytochrome C into the cytoplasm. This is a known means of converting pro-caspase-9 into its activated form, caspase-9, which can activate caspase-3 and trigger the irreversible apoptotic program. The bcl-2 protein family is the most important regulator of the intrinsic and apoptotic processes, and this family includes a pro-apoptotic member, such as Bax and such antiapoptotic members as bcl-2 and bcl-xL (Nahta and Esteva 2003).

The origin of sFas in patients with BC remains unclear. There are three possibilities. First, sFas may be derived from the tumor itself, corresponding to previous findings in tumor cell lines (Natoli et al. 1995). Second, sFas may be derived from peripheral blood lymphocytes (Kinipping et al. 1995). Third, the surrounding stromal tissue may produce sFas in response to tumor or immune activation (Natoli et al. 1995). In this study, we demonstrated that circulating plasma sFas concentration was increased in breast cancer patients compared to healthy controls and patients with benign breast masses, and also in cancer patients with advanced tumor stages (III, IV) compared to those with early stages (I, II), which indicates the important role of sFas in inhibiting apoptosis and helping tumor cells spread in advanced stages. Ueno et al. (1999) found that the median circulating sFas concentrations in primary and recurrent breast cancer patients were higher than in the normal female controls. Sheen-Chen et al. (2003) reported elevated mean value of circulating sFas in patients with advanced breast cancer than control group. In the present study, there was a positive correlation between sFas and some clinicopathological finding as fixation of the mass to chest wall and advanced tumor staging. In this respect, Ueno et al. (1999) reported that among primary cancer patients, significant correlation was found between sFas concentrations and menopausal status, a finding not confirmed in the present study. There are different hypotheses regarding the role of sFas in activated induced cell death and apoptosis. Some investigators report that during active apoptosis, released sFas may interact with FasL, inhibit cellular Fas/FasL interaction, and therefore inhibit apoptosis, but others postulate that sFas is released to exert a pro-apoptogen effect (Sahebari et al. 2010).

Bcl-2 is an antiapoptotic protein that maintains the integrity of mitochondria (Nahta and Esteva 2003). The result of this study demonstrated that plasma levels of bcl-2 was significantly increased in patients with benign and malignant breast masses compared to healthy controls and in breast cancer patients compared to those with benign breast masses. Also, this study revealed that levels of bcl-2 increased with advancing breast cancer stage with its highest level in stage IV. Gaballah et al. (2001) reported increased plasma levels of bcl-2 in 45 patients with metastatic breast cancer compared to healthy controls. They reported significantly higher levels in patients with positive ER. In this study, positive correlations between bcl-2 levels with factors that may affect outcome as progressive stage of the tumor, fixation to the breast and to chest wall, and also with other antiapoptotic factor as sFas were found. In this respect, Yang et al. (2009) reported that bcl-2 expression is highly associated with increased risk of local recurrence in patients with early stage of BC; meanwhile, others reported that high bcl-2 expression by tumor cells had no predictive value in breast cancer patients (Vargas-Roig et al. 2008). On contrary, correlation with good clinical and biological prognostic factors was shown by Jalava et al. (2000) who used immunocytochemical methods for bcl-2 determination. The association with good prognosis despite bcl-2’s antiapoptotic effect was explained by the fact that bcl-2 prolongs the cell cycle and slows cellular proliferation, as shown in an in vitro model, and the proliferative index in breast cancer specimens (Van Slooten et al. 1998). Bcl-2 blocks apoptosis via the mitochondrial pathway ((Nahta and Esteva 2003) and suppression of p21WAF1 expression (Bukholm et al. 1997).

Cytokines are low-molecular weight glycoproteins secreted by inflammatory and tumor cells, which can regulate cell functions. IL-8 and TNF-α are cytokines produced by mononuclear macrophages and endotheliocytes involved in activating and inducing T, B, and natural killer (NK) cells to target and phagocytosis pathogenic cells (Zhao et al. 2011). A number of studies showed that IL-8 can stimulate angiogenesis and cell migration and invasion in a variety of tumor cell types, including breast carcinoma cells, in autocrine and paracrine fashions, thereby increasing tumorigenicity and metastasis (Lang et al. 2002). In this study, plasma levels of IL-8 were significantly elevated in benign and malignant masses of breast than healthy controls and also in those with malignant than those of benign breast masses. Also, IL-8 plasma level was significantly elevated in patients with breast cancer stages III and IV than those with stages I and II and in those of breast cancer IV than those of stage III. In a study conducted by Benoy et al. (2004), serum IL-8 levels were higher in 69 patients with either operable or advanced breast cancer as compared to healthy women and they correlated directly with clinical stage of breast cancer. Elevated serum IL-8 levels were also associated with the occult cytokeratin-positive bone marrow cells, thus suggesting a correlation with poor prognosis (Benoy et al. 2004). Another study with 43 breast cancer patients suggested that tissue and plasma b chemokine RANTES (a member of the same family as IL-8) plays a role in carcinogenesis and that RANTES assay in tissue surrounding the tumor or in the tumor area after excision may help to predict unfavorable prognosis in breast cancer patients (Niwa et al. 2001). In this study, IL-8 was significantly positively correlated with antiapoptotic indices as sFas and bcl-2 indicating that causes of breast cancer are mutifactorial. Also, IL-8 was positively correlated with tumor stage and extent of the tumor spread evidenced by fixation to the breast and fixation of the mass to chest wall. De Larco et al. (2003) reported that IL-8 had a positive correlation with the invasive ability of breast cancer cells. On contrary, Balbay et al. (1999) showed no influence of IL-8 on the metastasis of prostate cancer cells and Lee et al. (2000) reported that IL-8 did not promote metastasis of ovarian cancer cells. Some reports have indicated that neutrophils attracted by IL-8 can release proteases, heparin, and enzymes in the process of moving to the region of a tumor, causing degradation of the extracellular matrix, resulting in tumor metastasis (Donskov et al. 2006). Lin et al. (2004) reported that expression of IL-8 is linked to status of ER, metastasis, and vimentin in breast cancer cells. ER expressions can downregulate IL-8 promotor activity. ER deletion has been consistently associated with tumor progression, recurrence, metastasis, and poor prognosis, but the biological mechanism is still unclear. In the present work, BC patients with ER positive status showed a insignificant negative correlation with plasma IL-8 level.

TNF-α, a cytokine mainly produced by activated macrophages exerts a major role in the immunoregulatory network. TNF-α is considered to be a mediator of NK cell function and can exert an important antitumor cytolytic activity (Gong et al. 2005). In this study, plasma levels of TNF-α were significantly elevated in benign and malignant breast masses than healthy controls and also in malignant than benign breast masses. Also, TNF-α plasma level was significantly elevated in patients with breast cancer stages II, III, and IV than those with stages I and healthy controls and showed progressive elevation with advanced breast cancer staging. Others reported elevated serum concentration of TNF-α in patient with breast carcinoma than healthy controls (Sheen-Chen et al. 1997). In this study, TNF-α was positively correlated with increasing tumor stage and extent of tumor progression. In this respect, Sheen-Chen et al. (1997) reported association between TNF-α and breast cancer progression. Gong et al. (2005) demonstrated that increased TNF-α promotes invasion and metastasis in ductal carcinomas in a scalar fashion. TNF-α secreted by tumor-related macrophages can enhance tumor invasion by increasing the expression of MMPs in breast carcinoma and vascular endothelial growth factor in the c-Jun N-terminal kinase, NF-kB signaling pathways (Hagemann et al. 2005), and activation of protein-1 that enhances angiogenesis (Szlosarek and Balkwill 2003). The positive correlation between TNF- α and antiapoptotic indices namely sFas and bcl-2 observed in the present study would indicate the role of these indices in tumor spread. Meanwhile, its correlation with IL-8 in a positive manner would indicate their effect in angiogenesis of breast tumors and their spread.

Increased oxidative stress and lipid peroxidation are implicated at various stages of carcinogenic processes. Earlier studies showed the involvement of reactive oxygen species (ROS) and reactive nitrogen species in cancer initiation and promotion (Haklar et al. 2001). NO is involved in the pathogenesis of many disorders including tumor development, immune responses, metastasis, and apoptosis (Suhendan et al. 2005). In this study, plasma levels of LPO and NO were significantly elevated in plasma of breast cancer patients than healthy controls and showed progressive elevation with advanced breast cancer staging. Meanwhile, NO level was elevated in patients with benign breast masses than healthy controls. In this respect, increased LPO in serum and tissues has been reported in breast cancer patients (Khanzode et al. 2004; Portakal et al. 2000). Thomsen et al. (1995) showed increased expression of both constitutive and inducible forms of NOS as well as increased NO production in breast cancer patients. Gaballah et al. (2001) reported elevated NO levels in metastatic breast cancer patients than healthy controls. Gönenç et al. (2006) demonstrated that in breast cancer, serum and tissue levels of NO were significantly increased compared to benign breast diseases. Meanwhile, others could not demonstrate either increased NO levels in malignant diseases (Liu et al. 1998) or their results supported NO role in antitumor immunity (Xie and Fidler 1998). Increased nitrate and nitrite levels in serum and tissue of breast carcinoma compared with those of benign breast diseases are explained by the increased expression of NOS activity by tumor cells (Thomsen et al. 1995). Enhanced production of NO in phagocytic cells may occur to protect them from oxidative stress (Gönenç et al. 2006). In the present study, there was a positive correlation between LPO and NO and some clinicopathological finding as fixation to the breast, fixation of mass to chest wall, and advanced tumor staging. Also, there were positive correlations between LPO and NO and antiapoptotic indices (sFas and bcl-2) and angiogenic factors (IL-8 and TNF-α). Damage to mammary epithelium by ROS can lead to fibroblast proliferation, epithelial hyperplasia, cellular atypia, and breast cancer (Portakal et al. 2000). Increased LPO associated with breast cancer development may offer a selective growth advantage to tumor cells over their surrounding normal counterparts (Rajneesh et al. 2008). A large body of clinical and experimental data suggests a promoting role of NO in tumor progression and metastasis. The possible mechanism(s) are (1) a stimulatory effect on tumor cell invasiveness caused by an upregulation of MMPs and a down regulation of their natural inhibitors, (2) a promotion of tumor angiogenesis and blood flow in tumor neovasculature, and (3) a suppression of host antitumor defense (Lala and Orucevic 1998). Pro-apoptotic effect of NO in cell often occurred when NO reacts with superoxide to produce the highly toxic peroxynitrite under condition of oxidative stress. Peroxynitrite anion toxicity not only relates to its ability to initiate lipid peroxidation, but it also releases of cytochrome C, disrupts mitochondrial transmembrane potential, depletes cellular glutathione, and damages DNA leading to cell death (Uchara et al. 1999). This was made clear in the present study as we found a significant positive correlation between serum levels of NO and LPO. Bcl-2 has been reported to counteract apoptotic cell death through multiple mechanisms, which mainly target mitochondrial events, but attention has been recently focused on its role in maintaining or augmenting cellular antioxidant defense capacity that involves antioxidant enzymes (e.g., catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase) and an antioxidant molecule glutathione (Jang and Surh 2003). In this respect, Lee et al. (2001) reported that bcl-2 raises cellular antioxidant defense status, but this is not necessarily reflected in decreased levels of oxidative damage to DNA and lipids. Increased oxidative stress is an important player in the initiation and progression of cancer, possibly through inflammatory responses. It has been reported that ROS stimulate NF-kB family of transcription factors involved in innate immune or inflammation responses. The activation of NF-kB upregulates the expression of many inflammation-related genes, including TNF-α, IL-6, IL-8, and vascular endothelial growth factor (Karin 2006).

Conclusions

Besides detecting apoptosis by invasive means in tumor tissue, apoptotic products can be quantified in the circulation. Although circulating apoptotic products usually lack organ and tumor specificity, they contribute in the assessment of disease extent or aggressiveness. The ease of drawing blood facilitates the serial measurement of circulating apoptotic markers to monitor tumor progress. Putting the results obtained by this study together, we could suggest that tumor cells of BC overexpress bcl-2 and sFas to secure their outgrowth and survival. However, this coincides with the activation of collection of physiologic regulatory mechanisms, such as increased IL-8, TNF-α, LPO, and NO, which try to stop tumor cells by inducing apoptosis. Meanwhile, excess NO and oxidative stress could damage DNA leading to gene mutations and cancer; meanwhile, NO also acts as angiogenic stimulators that help cancer spread. Increased expression of angiogenic cytokines as IL-8 and TNF-α which were measured in this study in BC stimulates cancer cell growth and contributes to locoregional relapse and metastases. These situations create an intracellular environment more favorable for macromolecule damage, mutations, and disease progression. It is possible that an improved understanding of interactions between signaling pathways, such as provided by the present study, will yield useful breast cancer therapies. Larger studies involving more patients with a longer follow-up are needed, together with a comparison of this ELISA method with immunohistochemical methods to more fully establish the utility of the apoptotic markers in breast cancer detection, staging, and intervention.

Limitation of the study

The small number of patients and study being cross-sectional rather than longitudinal are the major limitations of this study. The major cause was the limitation of the local research resources and budget to cover a few hundred patients required for proper sample size and an elaborate longitudinal and maybe interference study.

Conflict of interest

The author(s) declare that they have no conflict of interest.

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