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. Author manuscript; available in PMC: 2017 Jan 1.
Published in final edited form as: Mitochondrion. 2015 Dec 8;26:104–112. doi: 10.1016/j.mito.2015.12.001

Mitochondrial common deletion is elevated in blood of breast cancer patients mediated by oxidative stress

Hezhongrong Nie 1, Guorong Chen 2, Jing He 1, Fengjiao Zhang 1, Ming Li 1, Qiufeng Wang 1, Huaibing Zhou 1, Jianxin Lyu 1,*, Yidong Bai 1,*
PMCID: PMC4846287  NIHMSID: NIHMS747475  PMID: 26678158

Abstract

The 4,977 bp common deletion is one of the most frequently observed mitochondrial DNA (mtDNA) mutations in human tissues and has been implicated in various human cancer types. It is generally believed that continuous generation of intracellular reactive oxygen species (ROS) during oxidative phosphorylation (OXPHOS) is a major underlying mechanism for generation of such mtDNA deletions while antioxidant systems, including Manganese superoxide dismutase (MnSOD), mitigating the deleterious effects of ROS. However, the clinical significance of this common deletion remains to be explored. A comprehensive investigation on occurrence and accumulation of the common deletion and mtDNA copy number was carried out in breast carcinoma (BC) patients, benign breast disease (BBD) patients and age-matched healthy donors in our study. Meanwhile, the representative oxidative (ROS production, mtDNA and lipid oxidative damage) and anti-oxidative features (MnSOD expression level and variation) in blood samples from these groups were also analyzed. We found that the mtDNA common deletion is much more likely to be detected in BC patients at relatively high levels while the mtDNA content is lower. This alteration has been associated with a higher MnSOD level and higher oxidative damages in both BC and BBD patients. Our results indicate that the mtDNA common deletion in blood may serve a biomarker for the breast cancer.

Keywords: breast cancer; mtDNA mutations; 4,977 bp common deletion; MnSOD; oxidative stress

1. Introduction

Breast cancer is one of the most frequently diagnosed cancers in women worldwide (Kim et al., 2014), and it is also one of the leading causes of cancer-related deaths in Chinese women (Fan et al., 2014). The progression of breast cancer involves multiple genetic events. Tremendous progress has been made as a result of extensive research focusing on the nuclear genome. For example, mutations in the BRCA1 and BRCA2 genes encoded by the nuclear DNA (nDNA) are associated with a high risk of developing early onset breast cancer (Kwong et al., 2015). Similarly, mutations in TP53, PTEN (Suda et al., 2012) and CHEK1 and CHEK2 are also associated with increased susceptibility to breast cancer development (Sokolenko et al., 2015). In addition, increasing evidence suggests that mitochondrial function is severely impaired in various cancers including breast cancer (Deus et al., 2014; Neuzil and Moreno-Sanchez, 2013) due to genetic defects in the OXPHOS system (Chandra and Singh, 2011). Consequently, mtDNA mutations have been recognized as an important aspect of breast cancer occurrence and development (Nie et al., 2013; Shen et al., 2010; Shen et al., 2011; Yu et al., 2009).

Mitochondria are ubiquitous organelles and play important roles, including generating ATP through OXPHOS, producing ROS and initiating apoptosis (Desler et al., 2011). Mitochondria are the major intracellular source and also the most vulnerable targets of free radicals (Cui et al., 2012). Several studies have suggested that mitochondrial electron transport chain byproducts, i.e., free radicals (including ROS and RNS), may play multiple roles in tumor initiation, progression and maintenance (Brandon et al., 2006; Desouki et al., 2005; Li et al., 2012). Low levels of free radicals regulate cellular signaling and are essential in normal cell physiology (Matsubara et al., 2003). However, overproduction of free radicals may cause oxidation of a diverse array of cellular macromolecules, leading to alterations in normal cellular metabolism, signaling and function. Oxidative stress may occur if there is an imbalance between free radical production and antioxidant capacity (mainly comprising SOD, GPx and CAT) (Bresciani et al., 2015). The imbalance between oxidative species generation and antioxidant systems induced by both exogenous (diet and smoking) and endogenous estrogen factors have been implicated during the initiation, promotion and progression of breast cancer (Jerby et al., 2012; Vera-Ramirez et al., 2011; Yeon et al., 2011). The initial consequences of increased production of free radicals are changes in the structure of [the main/primary] biomolecules, including changes in nucleic acids (such as 8-OHdG) and lipids (such as MDA), which could lead to their dysfunction, while at the same time products derived from this damage are used as biomarkers of oxidative stress (Dayem et al., 2010; Gonenc et al., 2005; Sosa et al., 2013).

The mtDNA 4,977 bp or “common” deletion deletes between nucleotides 8,470 and 13,447 of the human mtDNA. This mutation removes all or part of the genes encoding four complex I subunits, one complex IV subunit, two complex V subunits and five tRNA genes, which are indispensable for maintaining normal mitochondrial function. It has been suggested that continuous generation of intracellular free radicals during OXPHOS is a major underlying mechanism for generating this deletion (Mandavilli et al., 2002; Santos et al., 2013), and such a deletion has been reported in several types of cancers (Chen et al., 2011; Dani et al., 2004; Tseng et al., 2006; Ye et al., 2008; Zhu et al., 2004). The common deletion in blood was first detected using the nest PCR method (Gattermann et al., 1995), and has since been found in intravital and postmortem blood (von Wurmb et al., 1998) and in different human blood cells (Meissner et al., 2000; Mohamed et al., 2004).

Somatic mtDNA deletions have been used as an indication of mtDNA oxidative damage (Meissner et al., 2008; Shen et al., 2010). In response to oxidative stress, an elaborate antioxidant system arises to mitigate the deleterious effects of ROS. MnSOD, a superoxide radical-scavenging mitochondrial enzyme, is crucial for protecting cells and tissues from oxidant injury and hypoxia by converting O2− to H2O2 (Candas and Li, 2014). Furthermore, recent studies have suggested that MnSOD may function as a tumor suppressor (Zhang et al., 2006). Human MnSOD is an 88.6-kDa tetrameric protein containing a Mn2+ ion associated with each subunit. Low MnSOD expression has been reported in several tumor cell lines compared to their noncancerous counterparts (Dhar and St Clair, 2012). Furthermore, MnSOD levels positively correlate with the in vivo tumor grade in breast cancers, particularly with the invasive and metastatic phenotypes of advanced breast cancers (Tsanou et al., 2004). However, reports that thyroid tumors, central nervous system tumors, gastric and colorectal carcinomas, and acute leukemia exhibited high MnSOD levels are conflicting (Park et al., 2013; Pica et al., 2015; Termini et al., 2015).

In this study, we aim to comprehensively evaluate the clinical relevance of the mtDNA common deletion, MnSOD, and oxidative damage in breast cancer, and try to identify novel mitochondrial biomarkers reflecting oxidative stress and cancer progression.

2. Materials and Methods

2.1 Clinical samples and DNA extraction

Blood and tissues of 107 patients with BC and 118 patients with BBD were collected at the Department of Pathology, First Affiliated Hospital, Wenzhou Medical University, Wenzhou, PR China, between May, 2007 and December, 2008. The internal controls were adjacent normal tissue (BC patients) and blood from the same subject before any chemotherapy, radiotherapy or pharmacotherapy. These patients were confirmed as BC or BBD by at least 2 senior pathologists. A total of 113 peripheral blood samples from age-matched healthy donors who had a cancer-free history, and had no other known diseases which could be associated with mitochondrial defects, were also collected at the Physical Examination Center of the same hospital from October, 2008 to December, 2008. All subjects gave their written informed consent, which was approved by the Wenzhou Medical University Ethics Committee, before enrollment. The carcinoma and paracarcinoma normal frozen tissues were dissected under microscopes and then DNA was extracted as described previously (Chen et al., 2011).

2.2 PCR amplification for mtDNA common deletion

Nest PCR was used for detecting the common deletion: the outer primers were used for amplification of a 495 bp product, while the inner ones were for a 358 bp product (Table S1). The amplification conditions for the outer primers were as follows: initial denaturation was at 95 °C for 5 min, followed by 35 cycles with denaturing at 94 °C for 20 s, annealing at 55 °C for 20 s, extension at 72 °C for 40 s, and a final extension at 72 °C for 5 min. The same amplification conditions were used for the inner primers, and 1 μl PCR product of the previous reaction was utilized as a DNA template while other conditions were the same except for the primers. The final PCR products were electrophoresed on a 2% agarose gel, and a 358 bp band was expected for patients exhibiting the common deletion (Figure S1A).

2.3 Quantification of common deletion level and mtDNA content

The common deletion level was measured by quantitative PCR (QPCR) on deletion products (Figure S1C) (Chen et al., 2011) and normalized by simultaneous measurement of overall mtDNA represented by COX-I fragment (Table S1) (Chen et al., 2011). Primers for construction of plasmids to generate templates for standard curves are also shown in Table S1. The probe for each PCR reaction was labeled with 5-FAM at the 5′ end and ECLIPSE at the 3′ end. The common deletion level was calculated using the ratio of the copy number of the common deletion to that of the COX-I fragment, while the mtDNA content was calculated from the ratio of COX-I fragment to the β-actin gene. DNA from HeLa cells was used as a control. The mtDNA content of breast tumor cells was presented relative to HeLa cells mtDNA content.

QPCR was carried out using an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems) in a 20 μl reaction containing 0.5 μM each of the forward and reverse primers, 0.1 pM for each probe (Cox-I and β-actin genes) and 10 ng total DNA sample. The amplification conditions were as follows: 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s and 58 °C for 30 s. Each sample was tested in triplicate and serial concentrations of standard plasmid were included for the standard curve.

2.4 Measurement of ROS production

For quantitation of ROS levels, fresh peripheral blood samples from 50 BBD patients, 46 BC patients and 45 healthy controls were collected, and plasma was separated, centrifuged and assayed with the Peroxide Assay Kit (Biomedical Research Service). This kit is based on the peroxide-mediated oxidation of Fe+2 to Fe+3 in the presence of xylenol orange dye, which can be conveniently quantified by spectroscopy at 595nm absorbance. The molar extinction coefficient of the xylenol orange-Fe+3 complex is 1.5 × 104cm−1M−1. Each sample was tested in triplicate, and the mean value was adopted for data presentation.

2.5 Determining the degree of oxidative mtDNA damage and lipid damage

The content of 8-hydroxy 2-deoxyguanosine (8-OHdG) in mtDNA was determined by a QPCR based technique as described in a previous report (Lin et al., 2008). One μl of sample DNA was treated with or without 1 U of hOGG1 in 1xNE buffer solution (20 mM Tris chloride, pH 8.0, 1 mM EDTA, 1 mM DDT, and 100 ug/ml of BSA) to a total volume of 10 μl at 37 °C for 1 h followed by 65 °C for 20 min (Lin et al., 2008). Then, the treated and non-treated DNAs were subjected to QPCR simultaneously. The Ct would increase from Ct1 to Ct2 if the mtDNA was harboring 8-OHdG (Lin et al., 2008). Using the equation for mtDNA quantification, we could calculate the total mtDNA templates under Ct1 and mtDNA templates without 8-OHdG formation under Ct2 to determine the degree of mtDNA integrity. The degree of oxidative mtDNA damage is defined as ΔCt, which is the difference between Ct1 and Ct2. The larger the ΔCt value, the more 8-OHdG the sample contains. Each reaction was performed in triplicate, and the mean value was adopted for data presentation.

For quantitation of oxidative lipid damage, plasma was de-proteinized by trichloroacetic acid precipitation. We mixed 0.1ml plasma with an equal volume of ice-cold 10% TCA, then incubated it on ice for 30 min. The samples were subjected to refrigerated centrifugation at 13,000g for 5 min, then the supernatant was recovered and combined with 0.1 ml of 2-thiobarbituric acid, and the mixture was incubated for 30 min at 95 °C. Samples were then cooled to room temperature, 0.2ml of n-butanol was added and the mixture was centrifuged for 3 min at 13,000g. The upper layer of each sample was removed, the absorbance at 532nm measured, and lipid peroxidation assays were performed as recommended by the manufacturer (Biomedical Research Service). MDA concentrations were calculated using a molar extinction coefficient of 1.56 × 105 cm−1M−1. Each reaction was performed in triplicate, and the mean value was adopted for data presentation.

2.6 Determination of MnSOD variation and expression

Eight pairs of primers were designed to amplify the 5′ flanking region, promoter region and 4 exons of the MnSOD gene (Table S2). The PCR conditions were set as: initial denaturation at 95 °C for 5 min, followed by 35 to 40 cycles of 94 °C, denaturation for 1 min, annealing at 51 °C to 63 °C for 1 min, extension at 72 °C for 2 min, and a final extension at 72 °C for 10 min. PCR products were then electrophoresed on a 2% agarose gel (Figure S1B). All PCR experiments included a negative control with no template DNA (double-distilled water). Then the products were purified using the Agarose Gel DNA Fragment Recovery Kit Ver.2.0 (TaKaRa, Japan) and subsequently sequenced on an ABI Prism 3730 sequence analyzer. These sequence results were compared with the updated consensus GenBank database.

Leukomonocytes were isolated from fresh peripheral blood with lymphocyte separation medium (Beyotime Biotech, Nantong, China) according to the manufacturer’s instructions. Cell lysis was performed by vigorous shaking for 10 min in lysis buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% NP40 and 0.1% SDS, supplemented with protease and phosphatase inhibitor cocktails I and II (Sigma, St. Louis, MO). Cell proteins were collected by centrifuging at 12,000 g at 4 °C for 10 min. Then, protein concentration was measured by the Bradford assay (Bio-Rad Laboratory, Hercules, CA). Lysates were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 50 μg aliquots were electrotransferred to a polyvinylydene fluoride (PVDF) membrane (Bio-Rad Laboratory, Hercules, CA). The membranes were then blocked in blocking buffer (5% milk in Tris-buffered saline containing 0.05% Tween 20) for 1 h at room temperature. The membranes were incubated overnight at 4 °C with different primary antibodies. The antibodies used for immunoblotting included anti-MnSOD and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoreactive bands were then detected by incubating with secondary antibody conjugated with horseradish peroxidase and visualizing using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA). Protein amounts were analyzed using ImageJ analysis software version 1.38e and normalized with their respective control. Experiments were performed at least three times and in duplicate.

2.7 Statistical analysis

All statistical analyses were performed using SPSS software (version 16.0) (SPSS Inc., Chicago, IL, USA). The frequency of the common deletion and MnSOD variation in all patients was tested by the Chi-square test or Fisher’s exact test. The mtDNA relative content and common deletion level were analyzed by Independence-Samples t-test. Hazard ratios (OR) of each parameter were presented with their 95% confidence intervals (95% CI) and all statistics were 2 sides. p<0.05 was considered significantly between groups.

3. Results

3.1 Detection of the common deletion in all patients

Nested PCR is a highly sensitive method for detecting large scale deletions even at low levels. To investigate clinical implications of the mitochondrial common deletion, we first examined cancer tissue, matched with adjacent normal tissue and blood samples from 107 BC patients, tissues and blood samples of 118 BBD patients and blood samples of 113 age- and sex-matched healthy controls, for this deletion using this method as described in the previous section. As shown in Figure 1, no significant differences were detected for the occurrence of the common deletion among cancer tissues, adjacent normal tissue and blood of BC patients. Similarly, we did not find differences between the tissues of BC and BBD patients for the occurrence of this deletion. However, interestingly, 51 out of 107 BC patients (47.66%) harbored the common deletion in their peripheral blood, which was significantly higher than that of BBD patients (10.17%) and healthy controls (8.85%) (p<0.001 for both). We can conclude that the common deletion occurring in blood was related to the progress of breast cancer development and thus may be a useful biomarker for diagnosis of breast tumors.

Fig. 1. Detection of the common deletion frequency in all patients.

Fig. 1

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BC: breast carcinoma, BBD: benign breast disease, Normal: healthy control.

Since the common deletion has been reported to accumulate during aging, and aging is also the biggest risk factor for cancer, we first divided our patients into 4 age groups to study the implications of aging for mtDNA defects. As shown in Table 1, within each age group, the BC patients had a higher occurrence of the common deletion in blood cells than the BBD patients and normal people. To explore what factors contribute to the increased occurrence of the common deletion in breast cancer, we further examined some clinical characteristics and other risk factors in these patients including body mass index (BMI), number of children, age, tumor stage, metastasis and expression of representative proteins that have been associated with breast cancer. We did not find any significant associations between the occurrence of the common deletion with BMI, number of children (Table S3), tumor stage, metastasis status and expression levels of ER, PR and Her-2 genes (Table 2). Interestingly, in BC patients, negative expression of TP53 and positive expression of Bcl-2 in the tumor tissue were associated with increased likelihood of detecting the common deletion (Table 2).

Table 1.

Detection of the 4,977 bp common deletion in blood in relation to age.

Age (Yr) Normal blood BBD blood BC blood P-value OR P-value# OR# P-value& OR&
N With CD Without CD N With CD Without CD N With CD Without CD
≤40 19 0 19 44 4 40 10 3 7 1.00 0.209 4.29
41–50 35 6 29 50 7 43 37 16 21 0.694 1.78 0.016 3.68 0.002 4.68
51–60 33 2 31 21 1 20 38 17 21 0.631 1.89 <0.001 12.55 0.001 16.19
>60 26 2 24 3 0 3 22 15 7 0.102 5.00 <0.001 25.71
Total 113 10 103 118 12 106 107 51 56 <0.001 9.38 <0.001 8.05
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P-value derived from Chi-square test for the detection of the 4,977 bp deletion in blood of BC patients when compared with the age factor.

P-value# derived from Chi-square between normal and BC patients in the same age group.

P-value& derived from Chi-square between BBD and BC patients in the same age group.

BBD: Breast benign disease; BC: Breast carcinoma; CD: Common deletion; OR: Hazard ratios.

Table 2.

Relationship between the frequency of the 4,977 bp deletion and clinicopathological parameters of BC.

Characteristic Blood Paracarcinoma Carcinoma
With CD Without CD P-value OR With CD Without CD P-value OR With CD Without CD P-value OR
Tumor Stage I 30 27 24 33 27 30
II 18 27 19 26 21 24
III 3 2 0.381 1 4 0.617 2 3 0.612
Metastasis + 14 25 15 24 17 22
− 37 31 0.065 0.469 29 39 0.672 0.841 33 35 0.622 0.820
ER# + 25 35 26 34 30 30
− 23 20 0.236 0.621 17 26 0.700 1.170 20 23 0.727 1.150
PR# + 25 36 26 35 29 32
− 23 19 0.168 0.574 17 25 0.828 1.092 20 22 0.994 0.997
Her-2# + 20 31 20 31 28 23
− 23 20 0.166 0.561 19 24 0.626 0.815 17 26 0.137 1.862
TP53# + 24 26 11 27 12 26
− 19 19 0.853 0.923 24 26 0.070 2.266 28 22 0.023 2.758
Bcl-2# + 30 30 30 30 36 24
− 16 18 0.784 1.125 12 22 0.168 1.833 11 23 0.010 3.136
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P-value derived from Chi-square test for detection rate of the 4,977 bp deletion in BC patients.

#

Incomplete data due to the loss of original clinical data.

ER: Estrogen Receptor; PR: Progesterone Receptor; Her-2: Human epidermal growth factor receptor-2;

TP53: Tumor Protein 53; Bcl-2: B-cell lymphoma 2.

3.2 The common deletion levels in BC and BBD patients

To further investigate the significance of the common deletion in breast cancer, we then measured its levels using QPCR in the patients carrying it. As shown in Table 3, although there was no significant difference between BC patients’ carcinoma tissues and BBD patients’ tissues (p=0.696), or between BC patients’ blood and adjacent non-tumor tissue (p=0.135), we found that the common deletion in blood of BC patients was markedly higher than in BBD patients (p<0.001). We also found that the 4,977 bp deletion level in the bloods was significantly higher than the carcinoma tissues of BC (p=0.032), while this common deletion level in the bloods was lower than that in the tissues of BBD patients (p=0.006). Moreover, in BC patients who carried the common deletion in both carcinoma and paracarcinoma tissues, the deletion level was always significantly lower in the carcinoma tissues (p=0.037).

Table 3.

mtDNA 4,977 bp deletion level in all breast tumor patients.

Characteristic BBD (n=54) BC (n=72)
Blood Tissue Blood Paracarcinoma Carcinoma
With CD 12 51 51 44 50
Minimum (E-05) 0.347 0.484 0.565 1.370 0.582
Median (E-05) 1.592 5.963 19.630 25.330 6.086
Maximum (E-05) 27.02 124.40 215.20 1126.03 182.10
Mean±SD (E-05) 4.16±7.47c, e 15.53±23.97a, e 34.76±47.59b, c, d 78.90±187. 23b, f 17.63±29.65a, d, f
95% CI (E-05) −0.58–8.91 8.79–22.27 21.37–48.15 21.98–135.82 9.20–26.05
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a

BC-c: BBD-t, p=0.696 ;

b

BC-b: p, p=0.135 ;

c

BC-b: BBD-b, p<0.001;

d

BC-b: c, p=0.032;

e

BBD-b: t, p=0.006;

f

BC-c: p, p=0.037.

Interestingly, the average and mean levels of deletion were negative correlated with age in BC (r=−0.3409, R2=0.0057) (Figure 2A) and BBD (r=−0.0216, R2=0.0008) (Figure 2B) patients’ blood and in BC (r=−7.4838, R2=0.1439) patients’ paracarcinoma tissues (Figure 2A), but were positive correlated with age in carcinoma tissues of BC patients (r=0.5606, R2=0.0408) (Figure 2A) and in BBD patients’ tissues (r=0.1378, R2=0.0034) (Figure 2B).

Fig. 2.

Fig. 2

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Correlation analyses between the 4,977 bp deletion level and age for A. BC patients and B. BBD patients.

3.3 Common deletion and mtDNA content

Alterations of mtDNA content have been widely reported in cancer patients (Chen et al., 2011; Mohideen et al., 2015; Shen et al., 2015), and mtDNA biogenesis can be induced upon stress (Dai et al., 2014). To investigate whether there was a correlation between mtDNA content and the common deletion in breast cancer, we plotted mtDNA copy numbers against common deletion levels in BC and BBD patients carrying this deletion. We found that with increased frequency of the mtDNA common deletion, there was no significant relationship with the mtDNA content in blood cells of BC (r=−8.8596, R2=0.0239) (Figure 3A) or BBD patients (r=−0.6165, R2=0.0097) (Figure 3B), or with the increased level of the common deletion in tissues of BBD patients (r=0.1288, R2=0.0006) (Figure 3B), in carcinoma tissues of BC patients (r=0.581, R2=0.0017) (Figure 3A) or in paracarcinoma tissues of BC patients (r=12.432, R2=0.0173) (Figure 3A).

Fig. 3.

Fig. 3

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Correlation analyses between the 4,977 bp deletion level and relative mtDNA content for A. BC patients and B. BBD patients. (b-blood, p-paracarcinoma, c-carcinoma, t-tumor.)

To examine the overall mtDNA content in BC and BBD patients, we measured mtDNA copy numbers in all patients, with and without the common deletion. We did not detect any significant difference in mtDNA content between cancer tissue and adjacent normal tissue (p=0.528). However, the mtDNA content in BC patients’ blood was significantly lower (p=0.028) than that of BBD patients, while the mtDNA content in both paracarcinoma tissues (p<0.001) and the carcinoma tissues (p=0.001) of BC patients was also significantly lower than in BBD patients (Table 4).

Table 4.

Relative mtDNA content for all patients with the 4,977 bp deletion.

Characteristic BBD (n=54) BC (n=72)
Blood Tissue Blood Paracarcinoma Carcinoma
With CD 12 51 51 44 50
 Mean±SD 1.43±1.19b 4.72±4.73c, d 0.77±0.83b 1.90±1.98a,c 2.17±2.12a,d
 Median 1.22 3.54 0.55 1.10 1.43
 95% CI 0.67–2.19 3.39–6.05 0.54–1.01 1.30–2.51 1.57–2.77
Without CD 42 3 21 28 22
 Mean±SD 1.18±1.13 2.21±1.76 0.52±0.29 1.50±1.79 3.09±4.82
 Median 0.63 2.01 0.50 1.09 1.65
 95% CI 0.83–1.53 −2.15–6.58 0.38–0.65 0.81–2.20 0.95–5.23
 P-value& 0.510 0.369 0.173 0.390 0.264
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a

BC-c: p, p=0.528;

b

BC-b: BBD-b, p=0.028;

c

BC-p: BBD-t, p<0.001;

d

BC-c: BBD-t, p=0.001;

&

between with CD and not.

3.4 Oxidative damage status in breast cancer patients

Since cancer cells are reported to be under increased oxidative stress (Deus et al., 2014; Neuzil and Moreno-Sanchez, 2013), and the mtDNA common deletion has also been suggested to result from oxidative damage (Santos et al., 2013), we first examined ROS production by direct biochemical measurements of cellular peroxide levels in representative patients with BC or with BBD and controls. As shown in Figure 4A, plasma peroxide levels were higher in both BBD and BC patients compared with healthy control subjects (p=0.0259 for BBD; p=0.0352 for BC).

Fig. 4.

Fig. 4

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A. Peroxide levels measured by a biochemical assay based on xylenol orange-iron complex formation; B. The mtDNA oxidative damage was evaluated by qPCR and defined as a ΔCt; C. Mean plasma levels of malondialdehyde (MDA) in patients (BBD and BC) and controls. (Sample sizes: control=45, BBD=50, BC=46; measured in blood samples.)

We then analyzed oxidative damage in mtDNA as indicated by the presence of 8-OHdG (Carioca et al., 2015) which was determined by a QPCR-based technique as described in a previous report (Lin et al., 2008). We detected significant increases in mtDNA damage in blood samples of both BC and BBD patients (Figure 4B).

Malondialdehyde (MDA) results from lipid peroxidation of polyunsaturated fatty acids, and the amount of MDA has been utilized to indicate the degree of lipid peroxidation (Gonenc et al., 2005). As shown in Figure 4C, the average and mean levels of MDA for BC patients were higher than those obtained for healthy subjects (p=0.0460).

3.5 Impaired MnSOD in BC patients

In response to oxidative stress, the antioxidant system is activated to mitigate the deleterious effects (Choudhari et al., 2014). MnSOD is a superoxide radical-scavenging mitochondrial enzyme and an important component of the cellular anti-oxidant system and altered levels have been implicated in breast cancer (Becuwe et al., 2014; Tsanou et al., 2004). We measured the expression of MnSOD in blood samples of BC and BBD patients (Figure 5), together with the control. We found that levels of MnSOD in both BC and BBD patients were significantly higher than in normal controls (p=0.0059 for BC and p=0.0292 for BBD).

Fig. 5.

Fig. 5

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Normalized expression of MnSOD in blood of age-matched healthy donors (ctrl), BBD and BC patients. (Sample sizes: Ctrl=45, BBD=50, BC=46; measured in blood samples.)

Because polymorphism of the MnSOD gene has been associated with increased risk of breast cancer, we then analyzed the 6 common MnSOD variations (Egan et al., 2003; Kostrykina et al., 2009) in our samples (Table 5). Among these 6 sites, we found that the C to G change at the −38 promoter region was significantly higher in BC patients compared with control individuals (p=0.002).

Table 5.

MnSOD variation sites.

site Genbank blood character BC (n=93) BBD (n=96) Control (n=197) Pa Pb
−2368 G G/A transition 13 13 31 0.697 0.622
−1402 G G/C transversion 27 25 46 0.298 0.614
−299 C C/A transversion 19 17 33 0.446 0.838
−38 C C/G transversion 64 59 98 0.002 0.059
106 A A/G transition 64 60 117 0.122 0.610
402 T T/C transition 29 26 44 0.105 0.371
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a

BC versus Control

b

BBD versus Control

4. Discussion

The mitochondrial DNA common deletion is one of the first described and most-studied mtDNA mutations, and it has been implicated in many human diseases (Dimberg et al., 2015; Li et al., 2015; Zhang et al., 2015). Multiple reports have associated the occurrence of this mtDNA common deletion with a wide range of cancer types including breast cancer (Lu et al., 2009; Nie et al., 2013). Investigations involving cell lines further implicated the common deletion in apoptosis and tumorigenesis (Lee et al., 2007; Wang and Lu, 2009).

The common deletion is more likely to be present in post-mitotic tissues with high energy demands, and its frequency increased with aging (Markaryan et al., 2008; Zhong et al., 2011). However, the presence of the common deletion in blood is inconclusive or controversial (Abnet et al., 2004; Dani et al., 2004; Mohamed et al., 2004; Upadhyay et al., 2009). Using a more sensitive detection method, we found that the common deletion is more likely to be present in BC patients’ blood compared with that of BBD patients and normal people (Figure 1, p<0.001 both).

As compared with deletion levels in the bloods, the carcinoma tissues from BC had lower level of 4977 bp deletion (p<0.032), but the tissues from BBD patients had higher common deletion level (p<0.006). For BC patients, the common deletion level in paracarcinoma tissues was significantly higher than that of the carcinoma tissues of BC patients (p=0.037). The high common deletion level in the blood of BC patients could be a consequence of the selective death of cancer cells, carrying higher common deletion levels, and the subsequent release of their DNA content including mtDNA from tumor tissues into the blood, while the decreased level of the common deletion in cancer tissue is likely the result of the same selection process.

Alterations in mtDNA content have been reported in many types of cancer, and multiple mechanisms have been proposed including dysregulation of mtDNA replication (Mambo et al., 2005; Shen et al., 2010; Sun et al., 2009; Tseng et al., 2006). We found that, compared with BBD patients, mtDNA content was lower in both blood and tissues of BC patients, possibly due to increased oxidative damage to the replication machinery (Agaronyan et al., 2015; Copeland and Longley, 2014).

Because the mtDNA common deletion has been suggested as an indicator of oxidative damage, we analyzed both the generation of ROS and the levels of oxidative damage in blood samples at the DNA and lipid levels. Our data showed that the oxidative stress and damage levels in breast tumor patients (BC and BBD) are higher than those in matched control individuals. The reason why BBD patients exhibited increased oxidative damage but did not show increased levels of the common deletion in blood samples is not clear.

One important regulatory mechanism in controlling oxidative stress/damage is the antioxidant system, including MnSOD. Among the 6 variation sites we analyzed in this study, −38C/G locates in the promoter region. Transcription factor database searches indicated that the −38C/G is within a binding site for both Sp1 and AP-2 (Xu et al., 2008). In cancer cells, high levels of MnSOD correlated with high levels of Sp1 and low levels of AP-2, whereas in transformed cells, low levels of MnSOD were associated with high levels of AP-2 and low levels of Sp1 (Xu et al., 2008). It is likely that polymorphism at the −38 position plays a modulatory role in expression of MnSOD.

Previously, correlations between MnSOD level and the frequency of mtDNA deletions have been reported in human liver and skin fibroblast cultures (Pitkanen and Robinson, 1996; Yen et al., 1994). Our results indicated that MnSOD levels in blood samples of BC and BBD patients were higher than in those of healthy controls, possibly as a reaction to increased oxidative stress (Polat et al., 2002; Yeh et al., 2005). However, such up-regulation was not enough to mitigate the oxidative damage induced by the enhanced ROS production.

Altogether, as illustrated in Figure 6, we reason that breast cancer cells are subjected to increased oxidative stress which induces activation of cellular anti-oxidant systems including MnSOD. When the oxidative stress level overcomes the cellular defenses, oxidative signaling will further activate the tumorigenesis cascade, and at the same time causes oxidative damage, including generation and accumulation of the mtDNA common deletion. Within the tumor, cell death occurs when the common deletion level reaches a threshold, and the DNA content will be released into the blood. If our reasoning is correct, then the mtDNA common deletion level in blood could serve as a useful biomarker for breast cancer.

Fig. 6.

Fig. 6

Open in a new tab

A model illustrating how the mtDNA common deletion play a central role in breast cancer patients.

Supplementary Material

1. Fig. S1 The method of detecting the mtDNA common deletion and MnSOD polymorphism.

A. The final PCR product was subjected to electrophoresis through a 2% agarose gel (M, DL2000 DNA marker; lane 1, negative control; lane 2, samples harboring the Δmt common deletion generate a 358-bp fragment; lane 3, positive plasmid control). B. Gel electrophorectogram of MnSOD amplification products through a 2% agarose gel (M, DL2000 DNA Marker; lane 1, negative control; lane 2 to 4, 5′ flanking region; lane 5, promoter region; lane 6 to 9, exons). C. Sequencing profile for confirming Δmt common deletion, the red box presents one of the 13bp direct repeats at positions 8470–8482 and 13447–13459.

2. Fig. S2 Standard curves for the three targets.

A. common deletion; B. Cox-I gene; C. β-actin gene.

3
4
5

Acknowledgments

This work was supported by the Zhejiang Provincial Natural Science Foundation of China (Grant No. LZ12H12001).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1. Fig. S1 The method of detecting the mtDNA common deletion and MnSOD polymorphism.

A. The final PCR product was subjected to electrophoresis through a 2% agarose gel (M, DL2000 DNA marker; lane 1, negative control; lane 2, samples harboring the Δmt common deletion generate a 358-bp fragment; lane 3, positive plasmid control). B. Gel electrophorectogram of MnSOD amplification products through a 2% agarose gel (M, DL2000 DNA Marker; lane 1, negative control; lane 2 to 4, 5′ flanking region; lane 5, promoter region; lane 6 to 9, exons). C. Sequencing profile for confirming Δmt common deletion, the red box presents one of the 13bp direct repeats at positions 8470–8482 and 13447–13459.

NIHMS747475-supplement-1.tif (1.4MB, tif)
2. Fig. S2 Standard curves for the three targets.

A. common deletion; B. Cox-I gene; C. β-actin gene.

NIHMS747475-supplement-2.tif (2.4MB, tif)
3
NIHMS747475-supplement-3.doc (36KB, doc)
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NIHMS747475-supplement-4.doc (35KB, doc)
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NIHMS747475-supplement-5.doc (28KB, doc)

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