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Keywords:

  • mtDNA;
  • minisatellite;
  • instability;
  • mutation;
  • colorectal cancer;
  • clinical value

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Most studies of mitochondrial DNA (mtDNA) mutations in colorectal cancer have used case-control and case-database comparisons without searching their clinical relevance. This study was to investigate colorectal cancer tissue-specific mtDNA mutations from 54 matched colorectal cancer and adjacent normal tissues and then to evaluate their clinical values. This study focused on analyzing control region including mtDNA minisatellites and coding regions. Cancer tissue-specific mtDNA mutations were found in over half of the patients (59%). The patterns of mtDNA mutations were substitution only (13%), mtDNA minisatellite instability (mtMSI) (20%) and both mutations combined (26%). mtMSI in colorectal cancer was mainly occurred in the 303 polyC (35%) and 16184 poly C (19%) minisatellite. mtDNA copy number and hydrogen peroxide level were significantly increased in colorectal cancer tissue. The amount of mtDNA large deletions was significantly decreased in colorectal cancer tissue compared with those from matched normal mucosa (p = 0.03). The activity of the mitochondrial respiratory chain enzyme complexes I, II and III in colorectal cancer tissues was impaired. mtDNA haplogroup B4 might be closely associated with colorectal cancer risk. The patient group harboring cancer tissue-specific mtDNA mutations showed larger tumor sizes (p = 0.005) and more advanced TNM stages (p = 0.002). Thus, mtDNA mutations in colorectal cancer might be implicated in risk factors that induce poor outcomes and tumorigenesis.

Colorectal cancer is the third most commonly diagnosed cancer in the world, but it is more common in developed countries. More than half of the people who die of colorectal cancer live in a developed region of the world (http://globocan.iarc.fr/). However, the incidence rate of colorectal cancer has continued to increase in many developing countries. The 2007 Annual Report of Korea Central Cancer Registry showed that the incidence rate of Korean colorectal cancer ranked third in males at 49.7 per 100,000 people; additionally the incidence rate reached the fourth highest level at 33.9 per 100,000 people in females. In particular, the incidence rate of colorectal cancer increased every year, and its mortality rate also tended to rise.1

A mitochondrion is a membrane-enclosed intracellular organelle in most eukaryotic cells, which contains its own DNA. Mitochondria generate most of the cell's supply of adenosine triphosphate (ATP), the major cellular energy source, and are involved in cell signaling, cellular differentiation and apoptosis, as well as the control of the cell cycle and cell growth. Human mitochondrial DNA (mtDNA) contains 16,569 base pairs (bp) and represents 0.1–1.0% of the total genomic DNA. A mammalian cell contains approximately 1,000 mitochondria, and each mitochondrion contains 2–10 DNA copies. Mitochondrial DNA has a 10- to 20-fold greater susceptibility to genetic mutation, because it does not contain introns in comparison to nuclear DNA, the mtDNA repair system is inefficient, and mtDNA has a higher exposure to reactive oxygen species (ROS) produced in the process of ATP synthesis.2, 3

There has been controversy about whether somatic mutations are primary or secondary to tumorigenesis. However, it has been recently suggested that mtDNA mutations are responsible for tumorigenesis (mitochondrial theory of cancer).4 Moreover, recent studies demonstrate that mtDNA mutations are involved in the development of metastatic potential in tumor cells by ROS overproduction.5, 6

Previous studies have shown that mtDNA mutations in human colorectal cell lines as well as in primary human colorectal tissues have been found to be present in both the noncoding and the coding regions of the mitochondrial genome.7–9 We coincidentally observed marked sequence variations in mtDNA among different normal donors.10 These observations indicated that a case-control study might have false results for the determination of pathologic mtDNA mutations directly related to diseases. Unfortunately, most mtDNA mutation studies in colorectal cancer have used case-control and case-database comparisons7–9 and additionally, a paucity of data are available on the clinicopathological values of colorectal cancer-related mtDNA mutations.

To avoid the potential pitfalls of case-control and case-database comparison studies and additionally to search for the clinical significance and implications of mtDNA mutations in colorectal cancer, this study analyzed an mtDNA control region, two coding regions and mtDNA minisatellites to check for mtDNA instability from both colorectal cancer tissues and corresponding normal mucosa of the same patients. We also comprehensively evaluated the clinicopathological data for their clinical importance and meanings.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Patient specimens and DNA extraction

This enrolled 54 patients with colorectal cancer who underwent elective surgery at the Chonnam National University Hwasun Hospital (Hwasun, Korea) from June 2004 to December 2010. All patients gave informed consent in accordance with the institutional review board policy prior to surgery. Patients' demographics of age, gender, preoperative carcinoembryonic antigen (CEA), tumor location, degree of differentiation and tumor node metastasis (TNM) stage were collected and analyzed.

Matched cancer tissue specimens and corresponding adjacent normal mucosa were collected from the same patients during the surgical operation (Supporting Information Table 1), and the obtained cancer and normal tissues were verified by histopathological examination. Additionally, peripheral blood samples were collected from 24 patients. The tumor tissue and adjacent normal mucosa were added to two test tubes containing 5 mL of phosphate-buffered saline (pH 7.4) on the day of surgery. Total DNA was extracted using QIAamp Blood Mini Kit (QIAGEN Korea, Seoul, Korea). The extracted DNA was dissolved in TE buffer (10 mM Tris–HCl, 1 mM ethylenediaminetetraacetic acid pH 8.0) and measured quantitatively using a spectrophotometer.

Direct mtDNA sequencing analysis

Our published protocols were used for direct sequencing analysis of the mtDNA control region, tRNAleu and the cytochrome b gene (CYTB).11 A set of designated primer pairs for the polymerase chain reaction (PCR) and direct sequencing of the mtDNA genes were provided in Supporting Information Table 2 and Supporting Information Figure 1. The sequencing analysis was performed using an automatic genetic analyzer (model ABL 3130XL; Applied Biosystems, Foster City, CA). The mtDNA sequences obtained were analyzed using MitoAnalyzer (http://www.cstl.nist.gov/biotech/ strbase/mitoanalyzer.html) with the Revised Cambridge Reference Sequence (http://www.mitomap.org/) and the Blast2 program (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) to determine mtDNA aberrations.

Determination of mtDNA instability using analysis of minisatellite markers

The three mtDNA minisatellite markers, such as 16189 poly C (16184CCCCCTCCCC16193, 5CT4C), 303 poly C (303CCCCCCCTCCCCC315, 7CT5C) and 514 (CA) repeat (514CACACACACA523, (CA)5 repeats) in mtDNA noncoding hypervariable regions (HV) 1 and 2 were used for checking mtDNA instability of colon cancer tissue and corresponding adjacent normal mucosa (Supporting Information Table 2). The method of analysis was size-based separation of amplified product by capillary electrophoresis based on our published protocol.11, 12 Thymine Adenine cloning for the confirmation of mtDNA minisatellite alterations was carried out according to a previously published protocol.11

Identification of mtDNA haplogroups in Korean patients with colon cancer

The mtDNA sequences of control and coding regions were assigned to haplogroups according to a classification previously proposed.13, 14 We compared the frequencies and distributions of haplogroups in this study with our published mtDNA sequence data10 and others15 from healthy Korean donors.

Quantification PCR for mtDNA copy number

Determination of mtDNA copy number was performed using our published methods.2 In brief, the method was as follows:[To generate a standard curve for quantification of mtDNA, the purified PCR products of CYTB and β-actin gene as an internal standard were inserted into pGEM-T easy vector, and Escherichia coli JM 109 cells (Promega) were transformed to obtain recombinant plasmids. A mixture of 25 μL containing 12.5 μL of 2× Quantitect SYBR green PCR master mix (Qiagen, Valencia, CA), 400 μM CYTB primers F14909 (5′-TACTCACCAGACGCCTCAACCG-3′) and R15396 (5′-TTATCGGAATGGGAGGTGATTC-3′), and 6 ng of total DNA was used for PCR with the Rotor-Gene real-time centrifugal DNA amplification system (Corbett Research, Sydney, Australia). For PCR, hot start reactions at 50°C for 2 min and at 95°C for 15 min were followed by 35 cycles of 20 sec at 94°C, 30 sec at 56°C, 30 sec at 72°C and a melting reaction with a decrease of 1°C per cycle between 72°C and 92°C. The mtDNA copy number was calculated using the following formula: {X μg/μL plasmid DNA/4419 (plasmid length) × 660} × 6.022 × 1023 = Y molecules/μL, where X represents the concentration of plasmid DNA and Y represents copy number. The mtDNA copy number was reported as the ratio of CYTB and β-actin gene (Supporting Information Fig. 2).

mtDNA large deletion

The mtDNA large deletion of 4,977 bp included ATPases 8 and 6, cytochrome c oxidase III and NADH dehydrogenase (ND)3, ND4, ND4L and ND5 regions. The primer pairs of int1F-3448 (5′-HEX-CCCTTCGCTGACGCCATA-3′) and int2R-3560 (5′-AGTAGAAGAGCGATGGTGAGAGC-3′) were used for generation of a fluorescently labeled 113 bp fragment of the undeleted mtDNA. Primers del1F-8395 (5′-HEX-CACCATAATTACCCCCATACTCCTTA-3′) and del2R-13494 (5′-GAGGAAAGG TATTCCTGCTAATGC-3′) were designed to flank the deletion breakpoints and were used to amplify a fluorescently labeled 123 bp product of deleted mtDNA. For PCR, a 50 μL of mixture containing DNA (50 ng), dNTPs (200 μM), primary primers (25 pmol), Taq DNA polymerase (2.5 U; TaKaRa LA Taq) and 10× buffer (5 μL) was used. An initial denaturation step at 96°C for 5 min was followed by 20 (int) cycles and 45 (del) cycles of 15 sec at 95°C, 20 sec at 60°C, 20 sec at 72°C and a 5 min final extension step at 72°C. After PCR was finished, a 1-μL aliquot of each PCR product and 0.5 μL of the internal size standard GS500 (Applied Biosystems) labeled with the fluorescent dye ROX (Applied Biosystems) were added to deionized formamide. Denaturation was performed at 96°C for 10 min followed by a cooling step at 22°C for 2 min. Denatured PCR products were separated by capillary electrophoresis using the ABI Prism 3130XL genetic analyzer (Applied Biosystems). When the run was completed, specific fragments were displayed as peaks in an electropherogram using the Gene Scan Analysis Software 3.1. Quantitative PCR was conducted with a Rotor-Gene real-time centrifugal DNA amplification system (Corbett Research) at a final reaction volume of 25 μL containing 2 μL of template DNA, 12.5 μL of 2× QuantiTect SYBR Green PCR master mix (Qiagen), DW, 10 pmol each of the forward primer del-lF (5′-CACCATAATTACCCCCATACTCCTTA-3′) and reverse primer del-2R (5′ GAGGAAAGGTATTCCTGCTAATGC-3′); for the other mixture, the primers were int-1F (5′-CCCTTCGCTGACGCCATA-3′) and int-2R (5′-AGTAGAAGAGCGATGGTGAGAGC-3′). After denaturation at 95°C for 15 min, the reaction mixture was cycled 45 times at 95°C for 15 sec, 58°C for 30 sec and 72°C for 90 sec. PCR was performed for 45 cycles (deletion primer-1F/2R) and 20 cycles (intact primer-1F/2R). The percentage of the 4,977 bp deletion was calculated according to the following formula: f (dmtDNA) = Admt/Amt × 1.9608CDmt/1.9613CDdmt × 123/113. The following are abbreviations and explanations for the aforementioned formula: f (dmtDNA), frequency of the 4,977 bp deletion; Admt, value of deletion-specific peak area; Amt, value of intact mtDNA-specific peak area; CDdmt, number of cycles necessary to detect the 4,977 bp deletion; CDmt, number of cycles necessary to detect intact mtDNA.

Determination of activity of mitochondrial respiratory chain enzyme complexes

Cell homogenates were diluted to 1 g/L total protein with 20 mmol/L potassium phosphate buffer (pH 7.2) before respiratory chain complex analysis. Assay conditions were based on previously published spectrophotometric methods.16

Quantitative determination of hydrogen peroxide in colorectal cancer tissue

To investigate the cause of colorectal cancer tissue-specific mtDNA mutations found in this study, colorectal cancer tissue and corresponding normal mucosa were measured for hydrogen peroxide concentration using our previously published protocols.2

Statistics

Spectra of mtDNA mutations were assessed with respect to patient gender and age, tumor size, preoperative CEA, degree of differentiation, tumor location and TNM stage using the chi-square test, Student's t test, and one-way ANOVA test. Significant levels were set at p < 0.05. All statistical analyses were carried out using SPSS for Windows, version 17.0.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Frequencies and types of heteroplasmic mtDNA mutations in colorectal cancer tissue

Overall, heteroplasmic mtDNA mutations which were only found in colorectal cancer tissue and not in corresponding adjacent normal mucosa from the same patient were observed in 32 patients (59%) in a set of analyses of the mtDNA control region, tRNAleu and CYTB obtained from colon cancer tissue and adjacent normal mucosa (Supporting Information Tables 3 and 4, and Figs. 1-I and 1-II). The pattern of these mtDNA mutations comprised substitution only (n = 7, 13%), mtDNA minisatellite instability only (mtMSI; n = 11, 20%) and combined substitution and mtMSI (n = 14, 26%).

Frequencies and types of heteroplasmic mtDNA mutations in colorectal cancer tissue

Colorectal cancer tissue-specific mtMSIs (dissimilarities in mtDNA instability patterns between corresponding adjacent normal mucosa and colorectal cancer tissue) were frequently found in our colorectal patients. The overall frequency of colorectal cancer tissue-specific mtMSI was 46% (n = 25). mtDNA minisatellite instability in colorectal cancer tissue mainly occurred in 303 polyC (n = 19, 35%) and 16184 poly C (n = 10, 19%) (Table 1). Additionally, the instability happened in the 514 (CA) repeat minisatellite (n = 4, 7%), in both 303 polyC and 16189 polyC (n = 3, 6%), in both 303 polyC and the 514 (CA) repeat (n = 2), and in both 16189 polyC and the 514 (CA) repeat (n = 1; Supporting Information Table 4, and Fig. 1-II).

Table 1. Summary and distribution of mtDNA minisatellite instability
inline image

Increase of mtDNA copy number in colorectal cancer tissue

The mtDNA copy number (ratio of CYTB and β-actin) from colorectal cancer tissue and corresponding adjacent normal tissue was 3,998 ± 1,975.8 and 3,329 ± 1,315, respectively (Fig. 2a). The mtDNA copy number was significantly increased in cancer tissue compared with either corresponding normal tissue (p = 0.048).1

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Figure 1. Sequencing chromatograms of mtDNA mutations (I) and minisatellite instability (II) in colorectal cancer. (I) Colon cancer tissue-specific mtDNA mutations were found in (a) HV1 (Patient No. 18) and (b) HV2 (Patient No. 30). These heteroplasmic mutations were not found in corresponding normal mucosa. (II) Gene scan analysis of (a) 303 polyC at np 303–315, (b) 16184 polyC at np 16,184–16,193 and (c) 514 (CA) repeat starting at np 514 revealed typical length heteroplasmic mutations (minisatellite instability) from Patient Nos. 10 and 17. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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thumbnail image

Figure 2. mtDNA copy number (a) and mtDNA large deletion (b). Increased mtDNA copy number in colon cancer tissue. mtDNA copy number from colon cancer tissues was significantly elevated compared with normal tissues (a). mtDNA large deletion (4,977 bp) occurred between two 13 bp direct repeats at positions 13,447–13,459 and 8,470–8,482. The amount of mtDNA large deletion was significantly decreased in colon cancer tissue compared with adjacent normal tissue (b).

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Decreased amount of mtDNA large deletion in colorectal cancer tissue

The mtDNA large deletion (4,977 bp) in colorectal cancer tissue and matched adjacent normal mucosa were quantified using gene scan and real-time PCR, which were developed in our laboratory. The amounts of mtDNA large deletion were significantly decreased in colorectal cancer tissue compared with those from matched normal mucosa (p = 0.03; Fig. 2b).

Impaired respiratory chain enzyme complex activity in colorectal cancer tissue

The activity of mitochondrial respiratory chain enzyme complexes in colorectal cancer tissues was impaired in comparison with those of the adjacent normal mucosa. Colorectal cancer tissues showed decreased enzyme activity in respiratory chain complexes I, II + III and no remarkable change of complex IV (Supporting Information Table 5).

Elevated hydrogen peroxide content in colorectal cancer tissue

Hydrogen peroxide was measured from colorectal cancer tissue and corresponding adjacent normal mucosa. The level of hydrogen peroxide was significantly elevated in supernatants from colorectal cancer tissue (45.1 ± 24.7 μM/mg protein) compared with those from adjacent normal mucosa (24.8 ± 8.6 μM/mg protein; p = 0.027) (data not shown).

Distribution of mtDNA haplogroups in Korean colon cancer patients

It is reasonable to compare our previous blood cell data, because the mtDNA analysis result of the blood and normal tissue are the same. Haplogroup D4 occurred most frequently in the Korean healthy donors as well as in colorectal cancer patients (Table 2). The general pattern and frequency of haplogroups in Korean colorectal cancer patients were similar to those of healthy donors. However, mtDNA haplogroup B4 disclosed an increased risk of colorectal cancer occurrence (odds ratio = 2.121; 95% confidential interval 1.507–2.986; p = 0.003; Table 2).

Table 2. Comparison of the frequencies of mtDNA haplogroups in healthy controls and patients with colon cancer
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Clinicopathological implications according to presence and type of mtDNA mutations in Korean patients with colorectal cancer

We first evaluated clinical pathological significance and implication of mtDNA alterations in colorectal cancer tissue according to mutation status by classifying patients into two groups: those with and without mtDNA mutations. The patient group harboring cancer tissue-specific mtDNA mutations showed larger tumor sizes (p = 0.005) and more advanced TNM stages (p = 0.002; Table 3). However, no significant differences between the two groups were found in the clinicopathological variables of age, sex, differentiation, tumor location, preoperative serum carcinoembryonic antigen level and depth of invasion (T stage). We next looked for the implications of each type of mtDNA mutation found only in colorectal cancer tissue. The patients were divided into three groups: substitution only, mtMSI only and both substitution and mtMSI. The patient group having both substitution and mtMSI showed larger tumor sizes (p = 0.012) and TNM stages (p = 0.017) compared with those from the groups with one mutation (substitution or mtMSI) and the no mutation group (Table 4).

Table 3. Clinicopathological significance of mtDNA mutations in colon cancer
inline image
Table 4. The correlation of patient clinicopathological demographics according to the types of mtDNA mutation
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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

This study investigated colorectal cancer-related mtDNA mutations through simultaneously analyzing mtDNA control, some coding and mtDNA minisatellite regions from both cancer tissue and corresponding adjacent normal mucosa, in addition to their clinicopathological implications in Korean patients with colorectal cancer. We found that cancer tissue-specific mtDNA mutations commonly occurred in the mtDNA control region, especially mtDNA minisatellite regions located in hypervariable segments of the mitochondrial genome. mtDNA alterations in colorectal cancer tissue further deteriorated mitochondrial function; the activity of mitochondrial respiratory chain enzyme complexes was impaired. Elevated ROS in colorectal cancer tissues might cause mtDNA mutations. These mutations subsequently induced mitochondrial dysfunction resulting in a vicious cycle. mtDNA alterations in colorectal cancer tissues might further impair a respiratory chain defect and increase the mtDNA copy number to compensate for the resultant ATP deficiency. During this perturbation, mitochondria might produce a large amount of ROS, which causes the progression of several diseases.2, 17

Overall, the frequency of heteroplasmic (cancer tissue-specific) mtDNA mutations was found in over half of participating patients (59%, 32/54). The patterns of mtDNA mutations in colorectal cancer included tissue substitution only (13%), mtMSI (20%) and both substitution and mtMSI combined (26%). Colorectal cancer tissue-specific mutations were frequently found as mtMSI. About half of all patients (46%) harbored mtMSI which mainly occurred in 303 polyC (35%) and 16184 poly C (19%). These results confirmed that the mtDNA control region (especially the displacement (D) loop region) is a hot spot for somatic mutations in colorectal cancer.7 Previous reports regarding the frequency of mtDNA mutations in colorectal cancer showed somewhat different results with a range of approximately 20–70%,8, 9 which might be caused by different methods, races and samples. Some chemical carcinogens bind preferentially to mtDNA as the major cellular target rather than nuclear DNA, and mtDNA mutation rates from tumors are higher than those from healthy tissues. These observations support the notion that somatic mtDNA mutations may be responsible for tumorigenicity. Recent studies have shown that mtDNA mutations can regulate tumor cell metastasis and one of the factors that induce metastasis.5, 6

Several models have been suggested for mtDNA copy number regulation. First, the requirement for ATP dictates the copy number of mtDNA. Second, the availability of nucleotides could regulate mtDNA replication. A third hypothesis proposes that mtDNA copy number is regulated by multiple replication origins.18 Our study showed that the mtDNA copy number was significantly increased in colorectal cancer tissue compared with corresponding normal mucosa. This finding might be explained by the excessive need for ATP as well as mitochondrial dysfunction resulting from mtDNA alterations in colorectal cancer tissue. However, almost nothing is known about the mechanism of mtDNA copy number monitoring. The mtDNA turnover rate and the proteins involved in the replication and degradation of mtDNA are obvious topics for future research.18

mtDNA large deletion (4,977 bp) mutations have been shown to be implicated in aging and carcinogenesis. The mtDNA large deletion increases in frequency in correlation with severity of dysplasia in Barrett's esophagus. However, in adenocarcinoma, the amount of this large deletion was remarkably depressed. Similar findings have been observed in thyroid, renal, hepatocellular, breast and nonmelanoma skin cancers, in which the 4,977 bp deletion is less abundant and less frequent in tumor tissue compared with adjacent nontumoral tissue. This study also showed similar findings to those observed in the aforementioned reports. The amounts of mtDNA large deletion were significantly decreased in colorectal cancer tissue compared with those from matched normal mucosa. These findings suggest an active selection pressure against the presence of mtDNA with the 4,977 bp deletion, and/or a population of tumor cells exist that do not undergo the typical aging response and subsequently form a tumor.19

mtDNA haplogroups have been associated with various cancers, metabolic diseases, aging and some neurodegenerative diseases. A previous report demonstrates that patients with mtDNA haplogroup M had an increased risk of breast cancer occurrence; additionally, the mtDNA haplogroup D4a was associated with an increased risk of thyroid cancer. On the other hand, no significant correlation between any mtDNA haplogroups and colorectal cancer was found.20 However, this study noted that mtDNA haplogroup B4 might be closely associated with the risk of colorectal cancer among Koreans. The number of patients in our study was too limited, and thus, extended investigation including a larger cohort would be warranted to reveal further the interaction between mtDNA haplogroups and the risk of colorectal cancer.

Although much knowledge has been collected concerning mtDNA alterations and their pathophysiological significance in colorectal cancer, less attention has been paid to the clinical values of mtDNA mutations, despite extensive evidence of mitochondrial involvement in tumorigenesis and disease progression. Recently, one study reported that the presence of the D-loop mutation might be a factor of poor prognosis and a factor of fluorouracil-based chemoresistance in colorectal colon cancers. Our study investigated clinicopathological values according to the existence of mutations, as well as specific mutation type. The patients having both substitution and mtMSI showed larger tumor sizes and advanced TNM stages compared with those from the patients with one type of mutation (substitution or mtMSI) or the no mutation group.

In conclusion, most colorectal patients harbored cancer tissue-specific mtDNA mutations as a type of substitution and minisatellite alteration, which were mainly caused by elevated levels of ROS in cancer tissue. mtDNA mutations might be associated with advanced stages; this is one of the risk factors that induce poor treatment outcome and prognosis in colorectal cancer.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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IJC_27375_sm_SuppFig1_2.ppt261KSupporting Information Figures
IJC_27375_sm_SuppTab1_5.doc548KSupporting Information Tables

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