Trastuzumab-based chemotherapy modulates systemic redox homeostasis in women with HER2-positive breast cancer

International Immunopharmacology 27 (2015) 8–14

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Trastuzumab-based chemotherapy modulates systemic redox homeostasis in women with HER2-positive breast cancer L.G.T. Lemos a, V.J. Victorino b, A.C.S.A. Herrera a, A.M.F. Aranome e, A.L. Cecchini e, A.N.C. Simão c, C. Panis d,⁎,1, R. Cecchini e,1 a

Bone Marrow Transplantation Center, National Cancer Institute, INCA, Rio de Janeiro, Brazil Faculty of Medicine, University of São Paulo, USP, São Paulo, Brazil Universitary Hospital, Department of Pharmacy, State University of Londrina, Londrina, Paraná, Brazil d Laboratory of Inflammatory Mediators, State University of West Paraná, UNIOESTE, Francisco Beltrão, Paraná, Brazil e Laboratory of Physiopathology and Free Radicals, Londrina State University, UEL, Londrina, Paraná, Brazil b c

a r t i c l e

i n f o

Article history: Received 9 January 2015 Received in revised form 7 April 2015 Accepted 17 April 2015 Available online 28 April 2015 Keywords: Trastuzumab Inflammatory status HER2 Oxidative stress Breast cancer

a b s t r a c t Trastuzumab is an immunotargeting therapeutic against breast tumors with amplification of the human epithelial growth factor receptor 2 (HER2). HER2 patients naturally exhibit disruption in the pro-oxidant inflammatory profiling; however, the impact of trastuzumab-based chemotherapy in modulating this process is still unknown. Here we determined the systemic pro-inflammatory profile of women diagnosed with HER2-amplified tumors, undergoing trastuzumab-based chemotherapy (TZ), and compared the results with that of healthy controls (CTR) and untreated patients with HER2-amplified breast cancer (CA). The plasmatic inflammatory profile was assessed by evaluating pro-oxidant parameters such as lipid peroxidation, total antioxidant capacity (TRAP), levels of advanced oxidation protein products (AOPPs), nitric oxide (NO), C-reactive protein (CRP), and total thiol content. Markers of cardiac damage were also assessed. Our findings showed increased NO levels in TZ than that in either CA or CTR groups. Furthermore, TZ augmented TRAP and reduced total thiol than that of the CA group. Our data also revealed that AOPP levels were significantly higher in the TZ than the CA group. AOPP and the MB fraction of creatine-kinase (CKMB) levels were positively correlated in TZ patients. These findings suggest that trastuzumab-associated chemotherapy can modulate the pro-inflammatory markers of HER2positive breast cancer patients to the levels found in healthy controls. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Despite significant advances in our understanding of cancer biology, breast cancer remains the most diagnosed neoplasia [1] and one of the leading causes of death among women worldwide [2]. Breast cancer is a heterogeneous disease in which several factors contribute to disease aggressiveness [3]. The amplification/overexpression of human epithelial growth factor receptor 2 (HER2) is an acquired genetic alteration that is well established as an indicator of poor prognosis in breast cancer [4], and it is found in approximately 20–30% of breast tumors [5]. HER2 is a potent oncogene that encodes a transmembrane tyrosine kinase receptor. The oncogenicity of HER2 depends on its dimerization with other HER family members; this interaction allows it to escape normal inhibitory regulation [6]. HER2-containing dimers have long halflives and exhibit strong and persistent signaling due to both slow ligand

⁎ Corresponding author at: Laboratory of Pathophysiology and Free Radicals, Department of General Pathology-Center of Biological Science, State University of Londrina, 86051-990 Londrina, Brazil. Tel.: +55 43 3371 4521; fax: +55 43 3371 4267. E-mail addresses: [email protected], [email protected] (C. Panis). 1 These authors equally contributed to this study.

http://dx.doi.org/10.1016/j.intimp.2015.04.037 1567-5769/© 2015 Elsevier B.V. All rights reserved.

dissociation and internalization [7]. Thus, HER2 excessively stimulates survival and mitogenic pathways [8,9], resulting in overall deregulated signaling [10], reduced apoptosis [11], prolonged cell survival [12], and increased metastatic potential [13]. The sustained signaling induced by HER2 amplification activates several cellular networks, which in turn affects the pro-oxidative inflammatory profiling [14]. The inflammatory process results from several systemic cellular processes, in which the generation of oxidative stress is an important component. Many cancer cells continuously experience oxidative stress due to the presence of significant amounts of reactive oxygen species (ROS) and an impaired antioxidant defense system [15]. Abnormal signaling in HER2-overexpressing cancer cells involving ROS are associated with alterations in PI3K-AKT, NF-κB, and p53 pathways [16,9,17]; all of these pathways are implicated in oxidative stress modulation. There is a crosstalk between inflammation and oxidative stress responses that depends on its intensity. In addition to its capability to cause DNA instability and cancer, high levels of oxidative stress can further promote disseminated damage to biological components such as lipids and proteins. This consequence can be measured in human plasma by evaluating the profiles of lipid peroxidation, protein oxidation and antioxidants consumption [15,40,43].

L.G.T. Lemos et al. / International Immunopharmacology 27 (2015) 8–14

Targeted immunotherapy using trastuzumab, a monoclonal antibody, was developed based on studies designed to identify strategies for HER2 inhibition. Trastuzumab therapy has been used successfully to treat HER2-amplified malignancies, in particular breast cancer [18]. Trastuzumab exerts its antineoplastic effects by blocking HER2 dimerization [19], thereby inhibiting aberrant receptor signaling [20]. As a consequence, ROS-generating pathways related to cardiac homeostasis seem to be attenuated, with deleterious effects [21,19]. Several studies have recently demonstrated that the chemotherapeutic agents employed for treating breast cancer can promote significant systemic pro-inflammatory changes [22–24]. In addition, a recent review by Zeglinski et al. [14] shows that most of the evidence regarding the impact of trastuzumab on the pro-oxidant cellular status are based on observations made in the context of cardiac cells that does not overexpress HER2 receptor. Although in vitro evidence suggests trastuzumab may induce pro-oxidative changes in breast cancer cells, the impact of this treatment in modulating the pro-inflammatory status of women with breast cancer is still unknown. In this study, we used highly sensitive methods to investigate the pro-inflammatory status of women with HER2-amplified breast tumors by evaluating the effect of trastuzumab-based chemotherapy on systemic oxidative stress parameters. Our aim was to characterize the modulation of the systemic inflammation and redox status induced by trastuzumab-based chemotherapy, as well as its impact on clinical features of breast cancer. 2. Patients and methods 2.1. Design of the study and selection of subjects This study was approved by the Research and Ethics National Council (CAAE 0009.0.268.000-07), and all participants provided informed consent. To determine a significant number of patients for this study, the following formula was applied: n0 ¼

Z 2  pð1−pÞ D2

n¼

n0 n0 1þ N

n0 = number scaled (384.16), Z = confidence level (1.96), p = probability (50%); D = margin of error (5%); n = sample size, and N = population size (N). According to the Instituto Nacional de Cancer estimative for breast cancer incidence in Brazil [25], 54 breast cancer cases are reported for 100.000 women in a region of 100.000 eligible women; on the basis of this estimate, significant sample size would be of 48 patients. In this study, 1008 women diagnosed with breast cancer were screened at the Londrina Cancer Institute, from February 2012 to June 2013. After application of the inclusion and exclusion criteria (described below), the cohort comprised 52 eligible patients. Since the amplification/overexpression of HER2 can be found in 20–30% of breast tumors, the minimal number of individuals in a HER2-amplified/overexpressing cohort is approximately 15 patients. In this study, a total of 57 women diagnosed with ductal infiltrative carcinoma of the breast bearing HER2 amplified tumors were enrolled to compose two groups: HER2 overexpressing cancer group formed by women bearing HER2 amplified tumors without any previous radio/chemotherapy (CA group, n = 24), and trastuzumab group, composed by women bearing HER2 amplified tumors which undergo the trastuzumab-based chemotherapy (TZ group, n = 33). The trastuzumab-based regimen consisted of 6 mg/m2 of intravenous infusion each 21 days with previous ACT scheme (4 cycles of doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 each 21 days during 12 weeks, followed by paclitaxel 175 mg/m2 each 21 days during 4 weeks, with attacking dosage of 8 mg/m2 of intravenous trastuzumab). Samples were collected during the cycle 8 of trastuzumab treatment, to

9

ensure that trastuzumab concentration in human plasma reached the steady-state [26]. Since our aim was to understand the overall impact regarding the scheme trastuzumab plus anthracyclins–taxanes scheme, we did not perform the analysis in a group of patients ongoing only the anthracyclins–taxanes scheme without trastuzumab. A control cohort was composed by 119 healthy volunteers, matched with the breast cancer cohort by ethnicity and age, without previous history of any type of cancer, chemotherapy, hormonal or antioxidant therapy, and chronic diseases. Women were excluded if they were currently smoking, had hepatic, cardiac or renal dysfunction, use of drugs, hypertension, diabetes and other eventual chronic conditions. Information on lifestyle and medical history were obtained at data collecting. Patients' clinical records were assessed to obtain patients information that included age at diagnosis, body mass index (BMI), chemotherapeutic regimen and tumor-node-metastasis classification, as well as data regarding tumor pathology (tumor size, histological grade, molecular receptors status and lymph nodal invasion). Nutritional habits of patients were similar to that of the control group. Samples were collected after signing informed consent, obtained from each patient or subject after full explanation of the purpose and nature of all procedures used. In all groups blood samples were obtained before chemotherapy starting and all patients presented four hour fasting before the plasma collection. The investigation was approved by the local ethical committee. Heparinized blood of all control participants was collected at Department of General Pathology, Londrina State University, PR-Brazil. Blood was centrifuged for red blood cells (RBC) and plasma obtainment. All analyses employing RBC were performed at the collection day and plasma aliquots were stored in −86 °C (Indrel Ultra Freezer) to further analysis. 2.2. Characterization of HER2 overexpression in breast tumoral tissue Tumor tissue sections (4 μM) were histologically analyzed for diagnosis by a pathologist. For immunohistochemical assay, the reaction was performed on 4 μM-thick paraffin-embedded sections from tumors by the labeled streptavidin biotin method using commercial kits with microwave accentuation. For each case, negative controls were performed on serial sections, whose were analyzed and classified as positive or negative [27]. Samples were considered as positive for estrogen (anti-human estrogen receptor α, clone 1D5, Dako) and progesterone (anti-human progesterone, clone PGR 636, Dako) receptors when at least 10% of the tumor cells nuclei were stained. For HER2 antibody (anti-human HER2-pY-1248, Clone PN2A), the positivity was considered when a strong membrane staining (3 +) was detected or FISH analysis amplification of HER2 in samples with moderate (2 +) membrane staining was detected. FISH analysis was performed in tissue sections using a Dako commercial kit (HER2 FISH PharmDXTM). HER2 interpretation followed the recommendations from Dako guidelines for breast cancer samples. 2.3. Pro-oxidative profiling of plasma The pro-oxidant status of plasma was characterized by measuring malondialdehyde (MDA) by high performance liquid chromatography (HPLC) [28], nitrite levels (NO) [29], advanced oxidation protein products (AOPP) [30], and lipid peroxidation evaluated by high-sensitivity chemiluminescence [31]. The antioxidant profile was determined by measuring the total antioxidant capacity of plasma (TRAP) by high sensitivity chemiluminescence, uric acid levels (URCA) and total thiol content [32]. 2.4. Analysis of the inflammatory status of the cardiac tissue Since trastuzumab caused important impact on the heart, we investigated the profiling of the cardiac damage markers creatine-kinase fraction B (CKMB), lactate-dehydrogenase (LDH), and C reactive protein

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(CRP). All samples were evaluated by a biochemical auto-analyzer (Dimension Dade AR Dade Behring, Deerfield, IL, USA), using Dade Behring® kits. Serum high-sensitivity C-reactive protein was measured using a nephelometric assay (Behring Nephelometer II, Dade Behring, Marburg, Germany). 2.5. Statistical analysis Analyses were conducted in duplicate sets and data expressed as means ± errors of the means. Parameters were compared by Mann– Whitney (non-parametric data) or Student's t test (parametric data). Spearman's correlations were also performed. All statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, San Diego, USA). A p b 0.05 value was considered significant and was represented in graphs by using capital letters, as follow: healthy controls × untreated cancer patients (marked as A), untreated cancer patients × trastuzumab-treated patients (marked as B) and healthy controls × trastuzumab-treated patients (marked as C). 3. Results Clinicopathological data of patients are presented in Table 1. All women with cancer were diagnosed as carrying ductal infiltrative carcinoma of the breast with amplification of HER2. The mean number of trastuzumab cycles at the moment of sample collecting was 8, which ensured the steady-state of this drug in plasma samples. The mean age at diagnosis was similar to all groups evaluated (47.77 years to control, 49.5 years to CA group and 48.12 years to TZ patients). Most of patients presented clinical early disease (TNM stages IIa and IIb). Analysis of the histological grade revealed a large percent of undifferentiated tumors in both cancer groups (CA and TZ groups). Pro-oxidant parameters measured in plasma are grouped in Fig. 1. MDA levels (Fig. 1a) were lower in TZ group when compared to both CA patients (154.9 ± 27.43 nM in TZ and 316.5 ± 29.32 nM in CA, p = 0.0004) and healthy controls (154.9 ± 27.43 nM in TZ to 345 ± 20.23 nM in CTR, p b 0.0001). CA versus controls did not show any significant difference (p = 0.6991). AOPP levels (Fig. 1b) were reduced in CA group when compared to controls (from 341.9 ± 18.90 μM in CTR to 194.5 ± 4.042 μM in CA, p b 0.0001) and augmented in TZ group in relation to CA patients (from 194.5 ± 4.042 μM CA to 333.9 ± 39.36 μM in TZ, p = 0.0119). AOPP levels in TZ group was similar to controls (p = 0.8409). NO in plasma (Fig. 1c) was decreased in CA group in relation to controls (from 17.58 ± 1.222 μM in CA to 27.26 ± 1.252 μM in CTR, p b 0.0001), while TZ patients exhibited higher levels than controls (32.77 ± 2.578 μM in CT plus TZ and 27.26 ± 1.252 μM in CTR, p = 0.0492) and CA patients (32.77 ± 2.578 μM in CT plus TZ and 17.58 ± 1.222 μM in CA, p b 0.0001). Two-way ANOVA analysis for

Table 1 Clinicopathological characterization of patients.

Number of patients Median age at diagnosis in years (range) Histological type Ductal Lobular Mixed Clinical disease Local Metastatic Histological grade 1 2 3

CA

TZ

n = 24 49.5 (29–72)

n = 33 48.12 (26–67)

100% none none

100% none none

100% –

90% 10%

0.8% 37.5% 61.7%

– 20% 80%

CA = women bearing HER2 breast cancer without any previous chemotherapy, TZ: women bearing HER2 breast cancer undergoing trastuzumab-based chemotherapeutic scheme.

lipid peroxidation curves (Fig. 1d) revealed that CA patients presented lower lipid peroxidation than controls (p = 0.0003). TZ group had enhanced lipid peroxidation profile when compared to both controls (p b 0.0001) and CA group (p b 0.0001). The antioxidant status is presented in Fig. 2. CA patients showed reduced antioxidant capacity (Fig. 2a) when compared to controls (278.5 ± 22.08 nM Trolox in CA and 2121 ± 173.0 nM Trolox in CTR, p b 0.0001). TZ enhanced TRAP levels in relation to untreated patients (1393 ± 143.4 nM Trolox in TZ and 278.5 ± 22.08 nM Trolox in CA, p b 0.0001). TRAP levels were similar between controls and TZ groups (p = 0.2533). When TRAP values were expressed in relation to uric acid levels (Fig. 2b) we still observed reduced antioxidant capacity in CA group (66.48 ± 5.269 nM Trolox/g × dL − 1 URCA in CA to 711.7 ± 45.23 nM Trolox/g × dL − 1 URCA in CTR, p b 0.0001). TZ presented reduced levels of TRAP/URCA in relation to controls (310.9 ± 32.00 nM Trolox/g × dL− 1 URCA in TZ and 711.7 ± 45.23 nM Trolox/g × dL−1 URCA in CTR, p b 0.0001) and higher levels in comparison to CA patients (310.9 ± 32.00 nM Trolox/g × dL− 1 URCA in TZ and 66.48 ± 5.269 nM Trolox/g × dL− 1 URCA in CA, p b 0.0001). TRAP levels expressed by thiol content (Fig. 2c) were similar to CTR versus CA group (2.759 ± 0.3198 nM Trolox/μM thiol in CA and 243.0 ± 17.46 nM Trolox/μM thiol in CTR, p b 0.0001), CA versus TZ (82.29 ± 12.10 nM Trolox/μM thiol in TZ and 2.759 ± 0.3198 nM Trolox/μM thiol in CA, p b 0.0001) and CTR versus TZ (243.0 ± 17.46 nM Trolox/μM thiol in CTR and 82.29 ± 12.10 nM Trolox/μM thiol in TZ, p = 0.0003). Table 2 shows the plasmatic levels of uric acid and thiol content in all evaluated groups. Uric acid did not vary in any group. Thiol content was significantly higher in CA patients than in controls (105.9 ± 5.046 μM in CA and 10.01 ± 0.401 μM in controls, p b 0.001). TZ displayed higher thiol content than controls (20.80 ± 2.053 μM in TZ and 10.01 ± 0.401 μM in CTR, p b 0.0001), but it was reduced in relation to CA group (20.80 ± 2.053 μM in TZ and 105.9 ± 5.046 μM in CA, p b 0.0001). Biochemical profile of cardiac markers and C reactive protein are shown in Fig. 3. C reactive protein (Fig. 3a) was augmented in CA group when compared to controls (6.495 ± 1.190 mg/dL in CA and 2.075 ± 0.2763 mg/dL in CTR, p b 0.0001). TZ had reduced levels of C reactive protein when compared to CA (2.380 ± 0.3079 mg/dL in TZ and 6.495 ± 1.190 mg/dL in CA, p = 0.0032). In comparison to controls, TZ patients exhibited augmented levels of C reactive protein (2.380 ± 0.307 mg/dL in TZ and 2.075 ± 0.2763 mg/dL in CTR, p = 0.0419). CKMB levels were significantly augmented in TZ group when compared to CA patients (18.24 ± 3.280 U/L in TZ and 1.591 ± 0.424 U/L in CA, p b 0.0001). LDH was observed at higher levels after TZ treatment (337.4 ± 30.03 U/L in TZ and 227.6 ± 27.10 U/L in CA, p = 0.0014). TZ patients also exhibited augmented levels of CKMB in relation to controls (18.24 ± 3.28 U/L in TZ and 2.847 ± 0.30 U/L in CTR, p b 0.0001), as well as higher LDH (337.4 ± 30.03 U/L in TZ and 165.4 ± 5.44 U/L in CTR, p b 0.0001). Spearman's correlation analysis was performed for all parameters, but only AOPP and CKMB (Fig. 4) showed a significant positive correlation in TZ patients (r = 0.4537, p = 0.0091). 4. Discussion The pro-oxidant status in breast cancer can induce two main types of response dependent on specific metabolites. Accumulating evidence shows that moderate levels of oxidative stress can have a proliferative effect on the cell cycle, while high levels are deleterious as they damage cellular structures [33–36]. In this context, the chemotherapeutic agents commonly used against breast cancer can induce ROS production as part of their antineoplastic mechanisms [37,38]. Because trastuzumab is suggested to have pleiotropic effects [39], we investigated the impact of trastuzumab-based treatment on the systemic pro-inflammatory status of breast cancer patients with HER2-amplified tumors. Since the pro-inflammatory status and the oxidative stress occurrence are linked, we performed several high-sensitivity analyses for detecting the

L.G.T. Lemos et al. / International Immunopharmacology 27 (2015) 8–14

a

b

p<0.001

p=0.0119

p=0.0004

800

800

AOPP levels ( µM)

M D A le v e ls ( n M )

p<0.001

1000

1000

11

600 400 200

600

400

200

0 CTR

CA

c

0

TZ

CTR

d

p=0.0492

2500

TZ

** ***

80 p<0.001 p<0.001

CA

CTR CA

2000

TZ

1500

RLU

NO levels ( µM)

60

40

CURVE STATISTICS * CTR x CA - p= 0.003 ** TZ x CTR - p<0.001 *** TZ x CA - p<0.001

1000

* 500

20

A 0 0

0 CTR

CA

5

10

15

20

25

30

Time

TZ

Fig. 1. Pro-oxidant parameters in plasma. Malondialdehyde (a), advanced oxidation protein products (b), nitric oxide (c), and lipid peroxidation profile (d) were evaluated as indicative of pro-oxidative status in plasma. Results are represented as individual dispersion data and medians, excepting for the lipid peroxidation profile. MDA = malondialdehyde; AOPP = advanced oxidation protein products; RLU = relative light unities. A p value b0.05 was considered significant. CTR: healthy control group, CA = women bearing HER2 breast cancer without any chemotherapy, TZ: women bearing HER2 breast cancer undergoing trastuzumab-based chemotherapeutic scheme.

molecular markers suggestive of the pro-inflammatory/oxidative processes. It is important to highlight that the aim of this study was to investigate the combined impact of trastuzumab plus anthracyclins– taxanes scheme. Therefore, in this study we did not include HER2 patients undergoing only anthracyclins–taxanes chemotherapy, without trastuzumab. We first evaluated the effect of trastuzumab on the plasmatic prooxidant profiling by investigating the level of lipid and protein oxidation. Trastuzumab-based treatment induced a global pro-oxidative

b p<0.001

-1

TRAP/URCA (nM trolox/gxdL URCA)

TRAP levels (nM trolox)

8000

6000

p<0.001

4000

2000

c

2000

TRAP/THIOL levels (nM trolox/uM thiol)

a

status, as evidenced by the high lipid peroxidation and enhanced formation of AOPP. Lipid peroxidation is a very complex chain of reactions. It results in the formation of several low-molecular-weight metabolites that form adducts with cellular structures such as protein and DNA [40]. Several factors can induce lipid peroxidation, including the NOderived metabolite peroxynitrite [41]. In this study, we observed high lipid peroxidation in association with elevated NO in plasma of patients undergoing trastuzumab-based therapy. The pro-oxidative status observed here indicates that trastuzumab-induced lipid peroxidation

p<0.001 p<0.001

1500

p<0.001

1000

500

CTR

CA

TZ

p<0.001

p=0.003

600

400

p<0.001

200

0

0

0

800

CTR

CA

TZ

CTR

CA

TZ

Fig. 2. Plasmatic antioxidant profile. Total antioxidant capacity of plasma (a), total antioxidant capacity of plasma in relation to uric acid levels (b), nitric oxide (c), and total antioxidant capacity of plasma in relation to thiol content (d) are presented in the plasmatic antioxidant profile. Results are represented as individual dispersion data and medians. A p value b0.05 was considered significant. CTR: healthy control group, CA = women bearing HER2 breast cancer without any chemotherapy, TZ: women bearing HER2 breast cancer undergoing trastuzumabbased chemotherapeutic scheme.

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L.G.T. Lemos et al. / International Immunopharmacology 27 (2015) 8–14 1000

Table 2 Uric acid and total thiol levels in plasma. CA

TZ

800

4.077 ± 0.2734 10.01 ± 0.4017

4.070 ± 0.1681 105.9 ± 5.046A

4.479 ± 0.3265 20.80 ± 2.053B,C

700

AOPP (µM)

Uric acid (mg/dL) Thiol content (μM)

900

CONTROL

Results are represented as mean ± standard errors of the mean. A p value b0.05 was considered significant and represented by using capital letters, as follow: healthy controls × untreated cancer patients (marked as A), untreated cancer patients × trastuzumab-treated patients (marked as B), and healthy controls × trastuzumab-treated patients (marked as C). CTR: healthy control group, CA = women bearing HER2 breast cancer without any chemotherapy, TZ: women bearing HER2 breast cancer undergoing trastuzumab-based chemotherapeutic scheme.

600 500 400 300 200 100 0

possibly due to the conversion of large amounts of plasmatic NO to peroxynitrite [42]. It is described that the stimulation of the HER2 receptor attenuates oxidative stress [39,49]. Therefore, inhibition of HER2 by TZ may affect the redox status of cells, triggering a pro-oxidant environment. In fact, the normal functioning of the neuregulin pathway promotes decreased ROS production, which in the presence of TZ is augmented, due to increased mitochondrial demand for ATP and NADPH oxidase activation under these conditions [14]. In this context, we assume that the impairment of cell survival by blocking HER2 causes increased oxidative stress that can further contribute to cell death, because high levels of oxidative stress can cause disseminated damage and adversely affect cell survival [15]. Furthermore, we found reduced levels of MDA, a secondary metabolite of the lipid peroxidation process, in patients receiving trastuzumabbased therapy. This indicated that the drug affects the formation of lipid peroxidation metabolites in plasma. The reduction in MDA levels may suggest the formation of other metabolites derived from the lipid peroxidation process, or even its consumption. Recent studies have

a

50

100

150

200

250

CKMB (U/L) Fig. 4. Correlation analysis between CKMB and AOPP levels in plasma from patients undergoing trastuzumab-based chemotherapy. Results are represented as individual dispersion data and evaluated by Spearman's correlation test. A p value b0.05 was considered significant. CKMB = MB fraction of creatine kinase, AOPP = advanced oxidation protein products. * indicates statistical significance (p b 0.05).

highlighted the anticarcinogenic potential of such molecules, including MDA itself [33–36]. Some patients from the TZ group presented levels of MDA similar to that found in the untreated cancer group, suggesting that the dynamics of its production and consumption can differ between individuals, even after TZ treatment. Thus, our findings suggest that trastuzumab is an important redox status modulator in patients, owing to its ability to increase NO-mediated lipid peroxidation and down modulate the production of lipid peroxidation-derived products, such as MDA.

p=0.0419

p<0.001

20

C Reative Proteins levels (mg/dL)

0

p=0.0032

15

10

5

0 CTR

b

CA

c

p<0.001

80

TZ

p<0.001

p<0.001

1000

800

60 LDH levels (U/L)

CKMB levels (U/L)

p=0.0014

40

20

600

400

200

0 CTR

CA

TZ

0 CTR

CA

TZ

Fig. 3. Cardiac-related inflammatory markers. Cardiac markers are represented here by C reactive protein (a), CKMB (b), and LDH (c) levels. Results are represented as individual dispersion data and medians. A p value b0.05 was considered significant. CTR: healthy control group, CA = women bearing HER2 breast cancer without any chemotherapy, TZ: women bearing HER2 breast cancer undergoing trastuzumab-based chemotherapeutic scheme. CKMB = MB fraction of creatine kinase, LDH = lactate dehydrogenase.

L.G.T. Lemos et al. / International Immunopharmacology 27 (2015) 8–14

Lipid and protein oxidations directly affect antioxidant defenses, since those processes mainly occur simultaneously [43]. Therefore, we investigated the systemic antioxidant status of patients undergoing trastuzumab therapy. Our findings demonstrated that trastuzumab restores the total antioxidant capacity of plasma of patients with HER2amplified breast cancer. Because uric acid and thiol content are the most abundant antioxidants evaluated by TRAP analysis [44], we also evaluated the antioxidant profile of plasma by correcting TRAP values in relation to these compounds. Our findings show that the ability of trastuzumab to recover the antioxidant capacity to control levels is independent of uric acid and thiol levels, suggesting that other antioxidants may be mobilized during treatment. TZ is known to cause cardiac toxicity, and the improvement of oxidative stress may affect such condition in the heart [14,39]. Despite the improved survival of patients treated with trastuzumab being welldescribed in the literature, side effects including cardiac toxicity have been reported [5,45]. In our study, approximately 75% of HER2 patients undergoing the trastuzumab therapy showed higher CKMB and LDH than the normal reference values, highlighting the occurrence of cardiac injury at the cellular level. These findings reinforce the physiological role of HER2 signaling in the maintenance of the integrity of the cardiac cells [46]. Oxidative stress in cardiac tissue may contribute to cardiac dysfunction following trastuzumab regimens, particularly when associated with doxorubicin chemotherapy [39]. In this study, we observed a positive correlation between CKMB and AOPP in the plasma. This suggests that protein oxidation may be related to alterations in cardiomyocyte permeability during trastuzumab treatment. In fact, blockade of HER2 impairs the ability of cells to activate survival pathways, which renders cardiomyocytes sensitive to ROS and leaves cellular structures sensitive to oxidation [14]. Furthermore, HER2 patients under chemotherapeutic regimens can suffer from cardiac toxicity induced by several oxidative stress-dependent mechanisms. These include impairment of the neuregulin–angiotensin I network, failure of mitochondrial electron transport, inhibition of MAPK/ERK signaling, and activation of the NADPH oxidase system; all these events are associated with reduced antioxidant capacity of cardiomyocytes [47]. Trastuzumab treatment also seemed to exert some anti-inflammatory effects on HER2 patients, reducing the levels of C-reactive protein in relation to untreated patients. In spite of this, trastuzumab improved the systemic AOPP formation in HER2 patients, which may indicate enhanced activation of the oxidative burst of neutrophils by the myeloperoxidase system, with subsequent hypochlorous acid formation [48]. This sustained protein oxidation possibly mediates alterations in cardiomyocyte permeability at the subclinical level, causing leakage and rise of CKMB levels in plasma as detected in this study. Our data indicate that TZ-based treatment is able to attenuate the systemic oxidative stress in cancer patients to normal levels, based on our evaluation of the plasma samples. Levels of plasmatic markers (CKMB/LDH), derived from the potentially damaged heart, were high after TZ chemotherapy. This suggests that although TZ recovers the systemic redox status in patients, mechanisms other than oxidative stress contribute to TZ-induced damage in the heart. In conclusion, our data show that trastuzumab modulates the systemic inflammatory status of women with HER2-amplified breast cancer by restoring the most of the pro-oxidative parameters to levels reported in healthy controls. Trastuzumab-based chemotherapy appears to improve the antioxidant capacity by elevating levels of available thiol and attenuation of systemic ROS-mediated events. There is the possibility for an interaction between protein oxidation and cardiac subclinical damage in this condition, however, our data underscores the need for long-term monitoring of subclinical markers of cardiac lesions in patients on trastuzumab regimens, even in the absence of overt clinical symptoms. Conflict of interest The authors declare that they have no conflict of interest.

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Acknowledgments The authors are grateful to Jesus Antonio Vargas and Instituto de Câncer de Londrina for the excellent technical assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2015.04.037. References [1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer statistics, Cancer J. Clin. 61 (2011) 69–90. [2] J. Lee, S. Giordano, J. Zhang, Autophagy, mitochondria and oxidative stress: crosstalk and redox signaling, Biochem. J. 441 (2012) 523–540. [3] A. Maxmen, The hard facts, Nature 485 (2012) S50–S51. [4] D.J. Slamon, G.M. Clark, S.G. Wong, W.J. Levin, A. Ullrich, W.L. Mcguire, Human breast cancer: correlation of relapse and survival with amplification of the HER-2/ neu oncogene, Science 235 (1987) 177–182. [5] H.M. Stern, Improving treatment of HER2-positive cancers: opportunities and challenges, Sci. Transl. Med. 4 (2012) 1–10. [6] J.W. Park, R.M. Neve, J. Szollosi, C.C. Benz, Unraveling the biologic and clinical complexities of HER2, Clin. Breast Cancer 8 (2008) 392–401. [7] I. Rubin, Y. Yarden, The basic biology of HER2, Ann. Oncol. 12 (2001) S3–S8. [8] C.Y. Chang, H.L. Chan, H.Y. Lin, T.D. Way, M.C. Kao, M.Z. Song, Y.J. Lin, C.W. Lin, Rhein induces apoptosis in human breast cancer cells, Evid. Based Complement. Alternat. Med. 2011 (2012) 1–8. [9] X.F. Wang, P.K. Witting, B.A. Salvatore, J. Neuzil, Vitamin E analogs trigger apoptosis in HER2/erbB2-overexpressing breast cancer cells by signaling via the mitochondrial pathway, Biochem. Biophys. Res. Commun. 326 (2) (2005) 282–389. [10] D. Harari, Y. Yarden, Molecular mechanisms underlying ErbB2/HER2 action in breast cancer, Oncogene 19 (2000) 6102–6114. [11] V. Aranda, T. Haire, M.E. Nolan, J.P. Calarco, A.Z. Rosenberg, J.P. Fawcett, T. Pawson, S.K. Muthuswamy, Par6–aPKC uncouples ErbB2 induced disruption of polarized epithelial organization from proliferation control, Nat. Cell Biol. 8 (2006) 1235–1245. [12] A.H. Kim, G. Khursigara, X. Sun, T.F. Franke, M.V. Chao, Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase, Mol. Cell. Biol. 21 (2001) 893–901. [13] T. Dittmar, A. Husemann, Y. Schewe, J. Nofer, B. Niggemann, K.S. Zänker, B.H. Brandt, Induction of cancer cell migration by epidermal growth factor is initiated by specific phosphorylation of tyrosine 1248 of c-erbB-2 receptor via EGFR1, FASEB J. (2002) 1823–1825. [14] M. Zeglinski, A. Ludke, D.S. Jasser, P.K. Singal, Trastuzumab-induced cardiac dysfunction: a “dual-hit”, Exp. Clin. Cardiol. 16 (3) (2011) 70–74. [15] B. Halliwell, Oxidative stress and cancer: have we moved forward? Biochem. J. 401 (2007) 1–11. [16] D.K. Biswas, Q. Shi, S. Baily, I. Strickland, S. Ghosh, A.B. Pardee, J.D. Iglehart, NF-kB activation in human breast cancer specimens and its role in cell proliferation and apoptosis, PNAS 101 (27) (2004) 10137–10142. [17] K.N. Schmidt, P. Amstad, P. Cerutti, P.A. Baeuerle, Identification of hydrogen peroxide as the relevant messenger in the activation pathway of transcription factor NF-kappaβ, Adv. Exp. Med. Biol. 387 (1996) 63–68. [18] J. Baselga, L. Norton, J. Albanell, Y.M. Kim, J. Mendelsohn, Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts, Cancer Res. 58 (1998) 2825–2831. [19] L.I. Gordon, M.A. Burke, A.T.K. Singh, S. Prachand, E.D. Lieberman, L. Sun, T.J. Naik, S.V.N. Prasad, H. Ardehali, Blockade of the erbB2 receptor induces cardiomyocyte death through mitochondrial and reactive oxygen species-dependent pathways, J. Biol. Chem. 284 (4) (2009) 2080–2087. [20] C.A. Hudis, Trastuzumab-mechanism of action and use in clinical practice, N. Engl. J. Med. 357 (2007) 39–51. [21] W. Tsang, G. Moe, Trastuzumab and heart failure, Cardiol. Rounds 8 (2007) 1–6. [22] C. Panis, A.C. Herrera, V.J. Victorino, A.M.F. Aranome, R. Cecchini, Screening of circulating TGF-β level and its clinicopathological significance in human breast cancer, Anticancer Res. 33 (2) (2013) 737–742. [23] C. Panis, L.G.T. Lemos, V.J. Victorino, A.C.S.A. Herrera, F.C. Campos, A.N. ColadoSimão, P. Pinge-Filho, A.L. Cecchini, R. Cecchini, Immunological effects of taxol and adryamicin in breast cancer patients, Cancer Immunol. Immunother. 61 (2012) 481–488. [24] C. Panis, A.C.S.A. Herrera, V.J. Victorino, F.C. Campos, L.F. Freitas, T. De Rossi, A.N. Colado-Simão, A.L. Cecchini, R. Cecchini, Oxidative stress and hematological profiles of advanced breast cancer patients subjected to paclitaxel or doxorubicin chemotherapy, Breast Cancer Res. Treat. 133 (2012) 89–97. [25] INCA, Instituto Nacional do Câncer: estimative of cancer incidence for 2012/2013 in BrazilAvailable in: http://www.inca.gov.br (Accessed Jul 19, 2013). [26] B. Leyland-Jones, K. Gelmon, J.P. Ayoub, A. Arnold, S. Verma, R. Dias, P. Ghahramani, Pharmacokinetics, safety and efficacy of trastuzumab administered every three weeks in combination with paclitaxel, J. Clin. Oncol. 21 (21) (2003) 3965–3971. [27] A.C.S.A. Herrera, C. Panis, V.J. Victorino, F.C. Campos, A.N. Colado-Simão, A.L. Cecchini, R. Cecchini, Molecular status is determinant on inflammatory status and

14

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35] [36]

[37]

L.G.T. Lemos et al. / International Immunopharmacology 27 (2015) 8–14 immunological profile from invasive breast cancer patients, Cancer Immunol. Immunother. 61 (2012) 2193–2201. V.J. Victorino, C. Panis, F.C. Campos, R.C. Cayres, A.N. Colado-Simão, S.R. Oliveira, A.C.S.A. Herrera, A.L. Cecchini, R. Cecchini, Decreased oxidant profile and increased antioxidant capacity in naturally postmenopausal women, Age 35 (2013) 1411–1421. C. Panis, T.L. Mazzuco, C.Z.F. Costa, V.J. Victorino, V.L.H. Tatakihara, L.M. Yamauchi, S.F. Yamada-Ogatta, R. Cecchini, L.V. Rizzo, P. Pinge-Filho, Trypanosoma cruzi: effect of the absence of 5-lipoxygenase (5-LO)-derived leukotrienes on levels of cytokines, nitric oxide and iNOS expression in cardiac tissue in the acute phase of infection in mice, Exp. Parasitol. 127 (2011) 58–65. M.A. Lozovoy, A.N. Simão, C. Panis, M.A. Rotter, E.M. Reiche, H.K. Morimoto, E. Lavado, R. Cecchini, I. Dichi, Oxidative stress is associated with liver damage, inflammatory status, and corticosteroid therapy in patients with systemic lupus erythematosus, Lupus 20 (12) (2011) 1250–1259. L. Pizzatti, C. Panis, G. Lemos, M. Rocha, R. Cecchini, G.H.M.F. Souza, E. Abdelhay, Labelfree MSE proteomic analysis of chronic myeloid leukemia bone marrow plasma: disclosing new insights from therapy resistance, Proteomics 12 (2012) 2618–2631. C. Panis, V.J. Victorino, A.C.S.A. Herrera, L.F. Freitas, T. De Rossi, F.C. Campos, A.N. Colado-Simão, D.S. Barbosa, P. Pinge-Filho, R. Cecchini, A.L. Cecchini, Differential oxidative status and immune characterization of the early and advanced stages of human breast cancer, Breast Cancer Res. Treat. 133 (2012) 881–888. M. Gago-Dominguez, E. Castelao, M.C. Pike, A. Sevanian, R. Haile, Role of lipid peroxidation in the epidemiology and preventions of breast cancer, Cancer Epidemiol. Biomarkers Prev. 14 (12) (2005) 2829–2839. F. Manello, V. Medda, G.A. Tonti, Protein profile analysis of the breast microenvironment to differentiate healthy women from breast cancer patients, Expert Rev. Proteomics 6 (1) (2009) 43–60. M. López-Lázaro, Dual role of hydrogen peroxide in cancer: possible relevance to cancer chemoprevention and therapy, Cancer Lett. 252 (1) (2007) 1–8. F. Manello, J.D. Raffetto, Matrix metalloproteinase activity and glycosaminoglycans in chronic venous disease: the linkage among cell biology, pathology and translational research, Am. J. Transl. Res. 3 (2) (2011) 149–158. G. Minotti, P. Menna, E. Salvatorelli, G. Cairo, L. Gianni, Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity, Pharmacol. Rev. 56 (2004) 185–229.

[38] J. Alexandre, C. Nicco, C. Chéreau, A. Laurent, B. Weill, F. Goldwasser, F. Batteux, Improvement of the therapeutic index of anticancer drugs by the superoxide dismutase mimic mangafodipir, J. Natl. Cancer Inst. 98 (4) (2006) 236–244. [39] E. Azambuja, P.L. Bedard, T. Suter, M. Piccart-Gebhart, Cardiac toxicity with antiHER2 therapies—what have we learned so far? Target. Oncol. 4 (2009) 77–78. [40] B. Halliwell, M. Whiteman, Measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and a what do the results mean? Br. J. Pharmacol. 142 (2004) 231–255. [41] R. Radi, J.S. Beckman, K.M. Bush, B.A. Freeman, Peroxynitrite oxidation to sulfhydryls, J. Biol. Chem. 266 (7) (2001) 4244–4250. [42] H. Rubbo, R. Radi, M. Trujillo, R. Telleri, B. Kalyanaraman, S. Barnes, M. Kirk, B.A. Freeman, Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation, J. Biol. Chem. 269 (42) (1994) 26066–26075. [43] B. Halliwell, Tell me about free radicals, doctor: a review, J. R. Soc. Med. 82 (1989) 747–752. [44] A. Ghiselli, M. Serafini, F. Natella, C. Scaccini, Total antioxidant capacity as a tool assess redox status: critical view and experimental data, Free Radic. Biol. Med. 29 (11) (2000) 1106–1114. [45] J.D. Hayes, D. Pulford, The glutathione S-transferase supergene family: regulation of GST and the contribution to the isoenzymes to cancer chemoprotection and drug resistance, Crit. Rev. Biochem. Mol. Biol. 30 (6) (1995) 445–600. [46] S.A. Crone, Y.Y. Zhao, L. Fan, Y. Gu, S. Minamisawa, Y. Liu, K.L. Peterson, J. Chen, R. Kahn, G. Condorelli, J. Ross Jr., K.R. Chien, K.F. Lee, ErbB2 is essential in the prevention of dilated cardiomyopathy, Nat. Med. 8 (5) (2002) 459–465. [47] L.P. Grazzette, W. Boecker, T. Matsui, M. Semigran, T.L. Force, R.J. Hajjar, A. Rosenzweig, Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes: Implications for herceptin-induced cardiomyopathy, J. Am. Coll. Cardiol. 44 (11) (2004) 2231–2238. [48] C. Yazici, K. Kose, M. Calis, S. Kuzuguden, M. Kirnap, Protein oxidation status in patients with ankylosing spondylitis, Rheumatology 43 (2004) 1235–1239. [49] V.J. Victorino, F.C. Campos, A.C. Herrera, A.N. Colado Simão, A.L. Cecchini, C. Panis, R. Cecchini, Overexpression of HER2/neu protein attenuates the oxidative systemic profile in women diagnosed with breast cancer, Tumor Biol. 35 (4) (2014) 3025–3034.