Overexpression of peroxiredoxin I and thioredoxin1 in human breast carcinoma

Abstract

Background: Peroxiredoxins (Prxs) are a novel group of peroxidases containing high antioxidant efficiency. The mammalian Prx family has six distinct members (Prx I-VI) in various subcellular locations, including peroxisomes and mitochondria, places where oxidative stress is most evident. The function of Prx I in particular has been implicated in regulating cell proliferation, differentiation, and apoptosis. Since thioredoxin1 (Trx1) as an electron donor is functionally associated with Prx I, we investigated levels of expression of both Prx I and Trx1.

Methods: We investigated levels of expression of both Prx I and Trx1 in breast cancer by real-time polymerase chain reaction (RT-PCR) and Western blot.

Results: Levels of messenger RNA (mRNA) for both Prx I and Trx1 in normal human breast tissue were very low compared to other major human tissues, whereas their levels in breast cancer exceeded that in other solid cancers (colon, kidney, liver, lung, ovary, prostate, and thyroid). Among members of the Prx family (Prx I-VI) and Trx family (Trx1, Trx2), Prx I and Trx1 were preferentially induced in breast cancer. Moreover, the expression of each was associated with progress of breast cancer and correlated with each other. Western blot analysis of different and paired breast tissues revealed consistent and preferential expression of Prx I and Trx1 protein in breast cancer tissue.

Conclusion: Prx I and Trx1 are overexpressed in human breast carcinoma and the expression levels are associated with tumor grade. The striking induction of Prx I and Trx1 in breast cancer may enable their use as breast cancer markers.

Figures

Figure 1

Expression Profiles of Peroxiredoxin I and Thioredoxin1 in 48 Major Human Tissues. The Human Major Tissue qRT-PCR array was used to determine transcript levels of Prx I (Figure 1A) and Trx1 (Figure 1B). For the human tissue array, tissues were selected from 48 individuals of different ethnicity. The y-axis represents the value of pg × 104 of DNA determined. Data were abtained using the comparative CT method with the values normalized to GAPDH levels and a standard curve. Details are in the "Materials and Method" section. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Prx I, peroxiredoxin I; qRT-PCR, quantitative real-time polymerase chain reaction; Trx1, thioredoxin 1.

Figure 2

Expression Profiles of Six Isoforms in the Peroxiredoxin Family in Major Human Tissues. The Human Major Tissue qRT-PCR array was used to determine transcript levels of Prx I-VI. Expression profiles of 26 tissues are displayed. The profiles of the 40 other tissues were deleted in this figure to simplify the display. Other details are in the legend of Figure 1. Abbreviations: Prx, peroxiredoxin; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 3

Increased mRNA Levels of Peroxiredoxin and Thioredoxin Families in Eight Cancer Tissues Compared with Normal Tissues. Cancer Survey qRT-PCR array was used to determine the transcript levels of Prx I-VI, Trx1, and Trx2 in breast, colon, kidney, liver, lung, ovary, prostate, and thyroid cancers. Samples in each of the eight cancer groups in the set of arrays consisted of three samples of normal tissue and nine samples of cancer tissues (cancer, phases I-IV) from different individuals. Data were analyzed using the comparative CT method with the values normalized to GAPDH levels. The y-axis represents the increase in the induction fold of the mRNA level of cancer tissue compared with the data from three samples of normal tissue. Error bar displays the range of standard error. Figure in inset is a scatter plot with individual values of the induction fold for Prx I depicted by each dot, the mean induction fold depicted by the longer horizontal line, and standard error depicted by the error bars (shorter horizontal lines) above and below the mean line. Clinicopathological information for each patient was provided by the supplier. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA; Prx, peroxiredoxin; qRT-PCR, quantitative real-time polymerase chain reaction; Trx, thioredoxin.

Figure 4

Predominant Expressions of Peroxiredoxin I and Thioredoxin1 mRNA in Breast Cancer Tissue. Data in Figure 4A and Figure 4C show the transcript levels of Prx I and Trx1, respectively. The BCRT II array (qRT-PCR) was used to determine the transcript levels of Prx I-VI, Trx1, and Trx2. Data were analyzed using the comparative CT method with the values normalized to β-actin level and expressed relative to controls. In parallel with each cDNA sample, standard curves were generated to correlate CT values using serial dilutions of the target gene. The y-axis represents the value of pg of DNA × 104. The induction fold data shown in Figure 4B and Figure 4D were obtained from the expression profiles in Figure 4A and Figure 4C, respectively. The BCRT II array consisted of five samples of normal breast tissue and 43 samples of breast cancer tissues from different individuals. Clinicopathological information for each patient was provided by the supplier. Values are reported as mean ± standard error. The t test was performed for levels of induction fold for Prx I versus other Prx isoforms (Figure 4B), and for Trx1 versus Trx2 (Figure 4D). The P values are represented by asterisks (** = P < .01, *** = P < .001). Abbreviations: BCRT II, Human Breast Cancer qRT-PCR Array II; mRNA, messenger RNA; Prx, peroxiredoxin; qRT-PCR, quantitative real-time polymerase chain reaction; Trx, thioredoxin.

Figure 5

Peroxiredoxin I and Thioredoxin1 mRNA Levels Associated with Grade of Breast Cancer. Data from the breast cancer groups using the Cancer Survey qPCR array (n = 9) and Breast Cancer qRT-PCR array I-V (n = 176) are displayed as a scatter dot plot with mean and standard error (Figure 5A). Data for induction fold for each cancer grade are represented as box-and-whisker plots with minimum and maximum. The t test was performed to compare induction fold between grade I and grade IV (Prx I, Figure 5B; Trx1, Figure 5C). The P values are represented by asterisks (** = P < .01). In addition, the Bonferroni test for multiple comparison was also performed. In this test, the P value was considered statistically significant if P < .1. The number of samples per grade and subdivided grade was distributed as follows: grade I, 37; grade II, 76 (IIA, 44; IIB, 32); grade III, 60 (IIIA, 32; IIIB, 9; IIIC, 19); and grade IV, 12. All samples in grade IV (n = 12) represent metastatic cancer. To investigate the association between induction fold and cancer grade, one-way ANOVA test for linear trend was performed between mean induction fold and subdivided cancer grades (Figure 5D). For Prx I, slope = 0.6217, P = .02; for Trx1, slope = 0.4497, P = .02. For both cases, linear trends were considered statistically significant if P < .05. Clinicopathological information for each patient was provided by the supplier. Abbreviations: ANOVA, analysis of variance; Prx I, peroxiredoxin I; qRT-PCR, quantitative real-time polymerase chain reaction; Trx1, thioredoxin 1.

Figure 6

Correlation Between Peroxiredoxin I and Thioredoxin1 mRNA Expressions in Breast Cancer. Data of induction folds of Prx I and Trx1 in breast cancer shown in Figure 5A are displayed as a scatter plot. Details are in the legend of Figure 5. Abbreviations: Prx I, peroxiredoxin I; Trx1, thioredoxin 1.

Figure 7

Western Analysis of Peroxiredoxin I and Thioredoxin1 Protein Expressions in Malignant and Normal Tissues. The total membrane and soluble protein lysates (15 μg) were loaded into reducing (Figure 7A and left side of Figure 7B) and nonreducing SDS-PAGE (right side of Figure 7B) and analyzed for protein expression. The sample information is described in Table 1. For example, N and C under the heading "Brain" are represented as BRN0 and BRC0 in Table 1, respectively. Figure 7B shows oligomerization for Prx I. Abbreviations: C, cancer (malignant); D, dimer; kDa, kilodalton; M, monomer; N, normal; Prx I, peroxiredoxin I; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel; Tet, tetramer; Tri, trimer; Trx1, thioredoxin 1.

Figure 8

Western Analysis of Peroxiredoxin I and Thioredoxin1 Protein Expressions in Malignant and Normal Tissues. Four samples each of normal and cancer tissue providing total membrane and soluble protein lysates (15 μg) were loaded into reducing SDS-PAGE (right side of Figure 8B) and analyzed for protein expression. The sets of three blots with one antibody (breast [BE], lung [LU], and ovary [OV]) were exposed on the same film at the same time. The sample information is described in Table 1. For example, N1 and C1 under the heading of "Breast (BE)" are represented as BEN1 and BEC1 in Table 1, respectively. Abbreviations: C, cancer (malignant); N, normal; Prx I, peroxiredoxin I; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel; Trx1, thioredoxin 1.

Figure 9

Western Analysis of Peroxiredoxin I, Peroxiredoxin II, Thioredoxin1, and Copper/Zinc Superoxide Dismutase Protein Expressions in Paired Samples of Malignant and Distant Normal Tissue Homogenates of the Same Patient. The total membrane and soluble protein lysates (15 μg) were loaded into reducing SDS-PAGE analyzed for protein expression. The patient information is described in Table 1. Cu/Zn SOD was included in this experiment as a positive control. Abbreviations: Cu/Zn SOD, copper/zinc superoxide dismutase; M, metastatic cancer; N, normal; P, primary cancer; Prx I, peroxiredoxin I; Prx II, peroxiredoxin II; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel; Trx1, thioredoxin 1.