J Gastroenterol DOI 10.1007/s00535-013-0845-7
ORIGINAL ARTICLE—ALIMENTARY TRACT
The prognostic significance of peroxisome proliferator-activated receptor b expression in the vascular endothelial cells of colorectal cancer Jin Zhou • Lie Yang • Yuan Li • Gunnar Arbman • Ke-Ling Chen Bin Zhou • Yong-Yang Yu • Cun Wang • Xian-Ming Mo • You Lu Zong-Guang Zhou • Xiao-Feng Sun
• •
Received: 10 April 2013 / Accepted: 4 June 2013 Ó Springer Japan 2013
Abstract Objective Currently, little is known regarding the role of peroxisome proliferator-activated receptor-b (PPAR b) in the vascular endothelial cells (VECs) of colorectal cancers (CRCs). The aim of this study was to investigate the relationship of PPAR b expression in the VECs of CRCs in terms of the prognosis and clinicopathological features of CRC patients. Design The expression and localization of PPAR b in the primary cancers and the matched normal mucosal samples of 141 Swedish CRC patients were analyzed in terms of its correlation with clinicopathological features and the
J. Zhou and L. Yang contributed equally to this study. J. Zhou L. Yang Y. Li K.-L. Chen B. Zhou Y.-Y. Yu C. Wang Z.-G. Zhou (&) X.-F. Sun Institute of Digestive Surgery and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China e-mail:
[email protected] G. Arbman Department of Surgery, Vrinnevi Hospital, Norrko¨ping, Sweden X.-M. Mo Laboratory of Stem Cell Biology and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China Y. Lu Department of Oncology, West China Hospital, Sichuan University, Chengdu 610041, Sichuan, China X.-F. Sun (&) Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linko¨ping University, ¨ stergo¨tland, Linko¨ping, Sweden Country Council of O e-mail:
[email protected]
expression of angiogenesis-related genes. This study also included 92 Chinese CRC patients. Results PPAR b was predominantly localized in the cytoplasm and was significantly downregulated in the VECs of CRC compared to that of the normal mucosa. The low expression levels of PPAR b in the VECs of CRC were statistically correlated with enhanced differentiation, early staging and favorable overall survival and were associated with the increased expression of VEGF and D2-40. The patients exhibiting elevated expression of PPAR b in CRC cells but reduced expression in VECs exhibited more favorable survival compared with the other patients, whereas the patients with reduced expression of PPAR b in CRC cells but increased expression in VECs exhibited less favorable prognosis. Conclusions PPAR b might play a tumor suppressor role in CRC cells in contrast to a tumor promoter role in the VECs of CRCs. Keywords Peroxisome proliferator-activated receptor-b Colorectal cancer Prognosis Vascular endothelial cells Angiogenesis
Introduction Peroxisome proliferator-activated receptor b/d (PPAR b/d), a ligand-activated transcription factor, belongs to the nuclear hormone receptor superfamily [1, 2]. PPAR b has been shown to play an essential role in the regulation of glucose homeostasis, skeletal muscle lipid metabolism [3], inflammatory response [4, 5], cellular differentiation and proliferation [6–8]. In recent years, PPAR b has been implicated in colorectal carcinogenesis, but whether it plays a promotive or inhibitive role remains highly controversial [9–14].
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To date, studies have shown that PPAR b elicits either no effect [15], attenuates or facilitates the pathogenesis of colorectal cancer (CRC) [16, 17], as reviewed previously [17, 18]. Our recent studies have indicated that PPAR b might facilitate the differentiation of CRCs while inhibiting their proliferation, and its expression in cancers significantly correlates with the favorable survival of rectal cancer patients [17, 18], indicating that its function is characteristic of a tumor suppressor. Ultimately, the precise role of PPAR b in the pathogenesis of CRC remains to be fully elucidated. Recent studies have reported that PPAR b might be involved in the tumor angiogenesis [19–21]. The expression of PPAR b is significantly upregulated in the vascular endothelial cells (VECs) of human pancreatic cancer tissues compared with normal pancreatic tissues [21]. Lewis lung carcinoma cells exhibited an increased hyperplastic microvascular structure and a decreased density of functional tumor microvessels after the knockdown of PPAR b [19]. The activation of PPAR b by the selective PPAR b ligand GW501516 can induce endothelial cells angiogenesis, including the increase of an angiogenesis-related gene vascular endothelial growth factor (VEGF) mRNA and the release of a VEGF peptide [22]. Recently, we observed the significant expression of PPAR b in the VECs of human CRCs by immunohistochemical assay (IHC) (unpublished data). Based on these observations, we hypothesized that PPAR b might play a role in the VECs of CRCs, which might be associated with the pathogenesis or development of CRC. Currently, little is known about this issue, and many fundamental questions need to be clarified: what is the expression pattern of PPAR b in the VECs of CRCs? What are the associations of its expression with the clinicopathological features and the prognosis of patients? To investigate the role of PPAR b in the VECs of CRCss, we examined the expression of PPAR b in the stromal VECs of CRC tissues and their respective matched normal mucosal samples of CRC patients. We analyzed the correlation of PPAR b expression in VECs and clinicopathological features, including the prognosis of CRC patients. Furthermore, we studied the association of the expression of PPAR b with that of angiogenesis-related genes including VEGF, particularly interesting new cysteine-histidine rich protein (PINCH), CD31 and D2-40 in CRC tissues. To the best of our knowledge, this study is the first that has investigated the expression of PPAR b in the VECs of CRCs and that found a correlation between its expression and the long-term prognosis of CRC patients.
Hospital and Vrinnevi Hospital in Norrko¨ping between 1982 and 2001. Each patient’s gender, age, tumor location, stage and differentiation were obtained from surgical and pathological records. All of the patients underwent tumor resection. Of them, five patients received preoperative adjuvant therapy (two patients with radiotherapy and three with chemotherapy). Informed consent was provided by all of the participants. Table 1 presents the characteristics of the patients and tumors including gender, age, location, stage, growth, and grade of differentiation. There were 75 male and 66 female CRC patients, with a mean age of 71 years (range 42–95 years) at the time of surgery. The primary tumors were distributed in the colon (n = 66) and rectum (n = 75). TNM staging classified 73 patients as stage I and II, and 68 patients as stage III and IV. The tumors comprised well- to moderately-differentiated adenocarcinomas (n = 94), and poorly differentiated adenocarcinomas (n = 47). Follow-up was conducted by matching all of the patients against the Swedish Cancer Register and the Cause of Death Register until 2010. The median follow-up period was 84 months (range 0–305 months). Information about the recurrence and overall survival of each patient was obtained from patient medical records. We conducted our study upon approval by the Institutional Review Board of the Linko¨ping University, Sweden. The expression of PPAR b was evaluated by immunohistochemistry (IHC) in the following set of colorectal Table 1 Characteristics of patients and tumors Characteristics
P value
Tumor VECs Positive (%)
Negative (%)
Gender Male Female
0.579 10 (48) 11 (52)
65 (54) 55 (46)
\70
7 (33)
53 (44)
C70
14 (67)
67 (56)
Colon
10 (48)
56 (47)
Rectum
11 (52)
64 (53)
6 (29)
67 (56)
15 (71)
53 (44)
Expansive
9 (43)
68 (60)
Infiltrative
12 (57)
46 (40)
8 (38) 13 (62)
86 (72) 34 (28)
Age (years)
0.354
Location
0.936
TNM stages I ? II III ? IV
0.021
Growth pattern
Materials and methods Patients and tissues This study included 141 patients with primary CRC. All of the patients were diagnosed at Linko¨ping University
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0.153
Tumor differentiation Good ? Moderate Poor
Pearson Chi-square test
0.003
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samples: 58 cases of carcinomas and accompanying normal mucosa and 83 cases of carcinomas. Distant normal mucosa samples were taken from the proximal margins of the resected histologically tumor-free and tumor-containing colorectal specimens. In the meantime, we collected an additional 92 primary human colorectal carcinoma samples. These tissue samples were collected between March and October 2009 during surgeries at the Department of Gastrointestinal Surgery, West China Hospital, Sichuan University, China. All of the patients provided their informed consent for participation in this study. The ethics committee of the medical center approved this protocol. All of samples were fixed in buffered formalin at 4 °C overnight, processed through graded ethanol solutions, and embedded in paraffin. Consecutive 4–6-lm tissue sections were cut from the paraffin blocks of each sample and mounted on polylysine-coated slides for IHC analysis. Cell lines The three human colon cancer cell lines HCT116, HT29 and SW480 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). The HCT116 cells were grown in McCoy’s 5A media (Genom BioMed Technology, Inc., Hangzhou, China), and the HT29 and SW480 were maintained in DMEM-F12, supplemented with 10 % fetal bovine serum (FBS), 100 IU/ml penicillin, 100 lg/ml streptomycin, and 1.5 mM L-glutamine in a humidified atmosphere of 5 % carbon dioxide at 37 °C. Immunofluorescence Immunofluorescence detection was performed to compare the localization of PPAR b by using a PPAR b antibody and a fluorescein isothiocyanate (FITC)-labeled secondary antibody with a DAPI-labeled staining solution. Briefly, 2.5 9 104 cells/mL were seeded into 6-well tissue culture plates and then incubated with both primary antibodies diluted in PBS overnight at 4 °C. After washing, the FITClabeled secondary antibodies were diluted in PBS for 1 h at room temperature in the dark. The slides were washed as previously described and the nuclei were counterstained with DAPI. Sections incubated with PBS in place of primary antibody served as negative controls. Images were acquired using a LEICA upright microscope, and photomicrographs taken at a 4009 magnification using a digital image acquisition system. Western blot analysis To evaluate protein levels in CRC tissues and cells and to validate the specificity of the anti- PPAR b antibody,
the HT-29, HCT116 and SW480 were grown to 90 % confluence in 6-well plates and rinsed once with phosphate-buffered saline (PBS) prior to harvesting. Cytoplasmic and nuclear proteins were separated and extracted from lysed, cultured HT-29, HCT-116 and SW480 cells using the NE-PER nuclear and cytoplasmic extraction kit (PIERCE Biotechnology, Rockford, IL, USA), according to the manufacturer’s protocol. The protein concentrations were determined using a Bradford protein assay. Lamin was used as a nuclear protein loading control, whereas b-actin was used as a total protein loading control. Western blot analysis was performed as previously described [16]. Briefly, fifty micrograms of protein from each sample was resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The samples were transferred onto a polyvinylidene difluoride (PVDF) membrane (0.2 lm, Bio-Rad) and detected by using a rabbit polyclonal antibody to PPAR b (1:1000; catalog no. s2709, Epitomics), rabbit anti-human b-actin antibody (1:1000, Millipore) or a rabbit anti-human lamin antibody (1:1000; Santa Cruz Biotech, Inc., Santa Cruz, CA, USA). The protein signals were analyzed using a goatanti-rabbit immunoglobulin (Ig)–horseradish peroxidase (POD) (Zhongshan Golden Bridge Biotech. Co., Ltd., Beijing, China) and a chemiluminescence reagent (Pierce, IL). The molecular mass of each visualized band was interpolated from a plot of log molecular mass versus distance migrated using kaleidoscope-prestained standards (Bio-Rad Laboratories). ImageJ Software (http://rsb.info.nih.gov/ij) was used to analyze band density. The band densities were calculated relative to the density of the b-actin bands. Immunohistochemical assay (IHC) Immunohistochemistry was performed on 5-lm sections from paraffin-embedded surgical specimens using either PPAR b or VEGF antibody. Sections known to exhibit high expression levels for PPAR b or VEGF were included in each run, serving as the primary antibody positive controls. In all of the staining procedures, the positive controls exhibited clear staining. For the negative control, PBS alone was substituted for the primary antibody to exclude possible false-positive responses due to non-specific secondary antibody background. Tissue sections were deparaffinized in xylene and rehydrated. For immunostaining, the sections were mounted on charged glass slides and boiled in 10 mM citrate buffer (pH 6.4) at 121 °C for 20 min for antigen retrieval and then processed by IHC. In general, after washing in PBS, the sections were incubated in 3 % H2O2-methanol for 15 min to block the activity of the endogenous peroxidases. After washing in PBS, the
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sections were incubated with power block (SP9000, Zhongshan Gold Bridge) for 30 min to reduce nonspecific background staining. The sections were incubated with appropriate primary antibody reaction, incubated at 4 °C overnight in IHC. After being washed in PBS, the sections were followed by incubation with a secondary antibody (Zhongshan Gold Bridge) for 40 min. The sections were then rinsed in PBS before reacting with streptavidin–biotin-peroxidase complex (SP9000, Zhongshan Gold Bridge) for 30 min each at 37 °C in IHC. The bound antibodies were visualized using 3,3-diaminobenzidine (DAB) as a peroxidase substrate. The sections were rinsed in water, counterstained with hematoxylin, dehydrated, and mounted in permanent mounting medium. Rabbit polyclonal antihuman PPAR b antibody (1:400, S2709; Epitomics) and rabbit polyconal anti-human VEGF (1:500, S1909-1; Epitomics) were used as the primary antibodies. Measurements of PPAR d expression by IHC The IHC slides were examined independently in a blinded manner by two investigators (Zhou J and Yang L) without clinicopathological information or biological data. Each investigator estimated the proportion of cells stained and the intensity of staining within the whole sections. The results of the cytoplasmic PPAR b staining in the epithelial cells in the normal mucosa, VECs or tumor cells are expressed as a percentage of positive cells, and the intensity of staining was estimated on a scale from 0 to 3 (lowexpression, weak staining: exhibited as light yellow; moderate staining: exhibited as yellow brown; and strong staining: exhibited as brown). The proportion of staining was scored as 0 (0–9 %), 1 (10–39 %), 2 (40–69 %) or 3 (70–100 %), according to the proportion of positively stained areas in relation to the whole carcinoma area. The percentage of cells at each staining intensity was multiplied by the corresponding intensity value to obtain an immunostaining score that ranged from 0 to 12. The scores were combined to obtain an overall mean score. If there was a discrepancy in the individual scores, then the both investigators reevaluated the slides together to reach a consensus prior to combining the individual scores. To avoid an artificial effect, the cells on the margins of the sections and in areas exhibiting poor morphology were not counted. For statistical analysis, the negative or weakly stained cases were considered as low expression, and the moderate or strongly stained cases were considered as high expression. Furthermore, as we have shown that the elevated expression of PPAR b in tumor cells was related to the favorable survival of the rectal cancer patients examined in our previous study [16], here we combined the expression of PPAR b in tumor cells and VECs to analyze its correlation with the prognosis of the patients. We divide the cases into
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four groups based upon PPAR b expression in the tumor cells and VECs as follows: high expression in the tumor cells with low expression in the VECs [i.e., T(high)V(low)], T(high)V(high), T(low)V(low) and T(low) V(high). Evaluation of PINCH, D2-40 and CD31 The data for PINCH (n = 80), D2-40 (n = 116) and CD31 (n = 116) of primary CRCs determined by IHC, were taken from previous studies performed with the same cases used in the present study at our laboratory [23, 24]. Statistical analysis All statistical analyses were performed using the STATISTICA software package (version 8.0; STATSOFT Inc. Tulsa, OK). The data were expressed as the mean ± standard error of the mean (SEM). The intensity of PPAR b, VEGF, PINCH, CD31 and D2-40 expression or clinicopathological factors were analyzed using the Chi-square test, and the other data were analyzed using one-way ANOVA. The Kaplan–Meier product-limit method was applied to graph survival. The log-rank test was used to compare the equality of the two curves. Multivariate analysis was performed with the Cox proportional hazards model using hazard ratios (HRs) and 95 % confidence intervals (CIs) to evaluate independent prognostic factors. Differences with p \ 0.05 were considered as statistically significant.
Results The anti-PPAR b antibody is highly specific and detects cytoplasmic PPAR b in colon cancer cell lines To examine the specificity of the anti-PPAR b antibody, we performed immunofluorescence and Western blot to detect PPAR b protein in three different-staged colon cancer cell lines HT 29 (stage 2), HCT116 (stage 3), and SW480 (stage 4). By immunofluorescence, PPAR b exhibited a prominent cytoplasmic localization in the three cell lines (Fig. 1a). Further, we fractionated the cytoplasmic and the nuclear proteins of the colon cancer cell lines and examined the expression of PPAR b protein by Western blotting analysis. As shown in Fig. 1b, each of the electrophoretic lanes had a clear band at the expected position for PPAR b, as well as the band of b-actin. All of the three cell lines exhibited significantly increased cytoplasmic PPAR b than nuclear PPAR b, and there were no significant changes in PPAR b expression between the stage 2 to stage 4 cell lines.
J Gastroenterol
Fig. 1 PPAR b expression in colorectal cancer cell lines HT-29, HCT-116, and SW480. A By immunofluorescence (IF), PPAR b was predominantly detected in the cytoplasm (black arrow) of the CRC cells, with minor staining in the nuclei. All of the images were acquired at a magnification of 9400. B Western blot analysis showed that the expression of PPAR b was significantly increased in
cytoplasmic compared to the nuclear compartment of the CRC cell lines; C Quantitative analysis showed that the altered localization of PPAR b protein was identical to that of the IF staining. The data shown represent at least three independent replicates of each experiment
The expression of PPAR b in VECs and its relationship with the clinicopathological features of CRC patients
the patients. As shown in Table 1, the tumors with reduced expression of PPAR b in the VECs exhibited a significant correlation with earlier stages (stage I ? II) (55.8 %, 67/120) in contrast to those with elevated PPAR b expression (28.5 %, 6/21; P = 0.021) and comprised better-differentiated cases (good ? moderate) (71.7 VS. 38.1 %; P = 0.003). There was no significant correlation of PPAR b expression in the VECs of CRCs with gender, age, tumor location or growth pattern (P [ 0.05).
By IHC analysis, we detected predominantly cytoplasmic PPAR b in the VECs, tumor cells and normal mucosal epithelial cells, with a little nuclear localization. In the following analyses in this study, we measured and present the cytoplasmic staining of PPAR b in the tumor cells and VECs. We compared the PPAR b expression in the VECs of CRCs (n = 141) with that of normal mucosa (n = 58). As shown in Fig. 2, the cancers exhibited a significantly increased frequency of reduced PPAR b expression (85.1 %, 120/141) in the VECs compared to the normal mucosa specimens (8.6 %, 5/58) (P \ 0.001). We further compared the PPAR b staining in 58 cases with the matched cancers and normal mucosa. Consistently, there was a significantly higher frequency of cases with reduced PPAR b expression in the VECs of CRCs (86.2 %, 50/58) compared to that of the normal mucosa specimens (8.6 %, 5/58; P \ 0.001). We analyzed the correlation of PPAR b expression in the VECs of CRCs with the clinicopathological features of
The expression of PPAR b in the VECs of CRCs is negatively associated with the prognosis of CRC patients We analyzed the correlation of PPAR b expression in the VECs of CRCs with the prognosis of patients by Kaplan– Meier analysis. As shown in Fig. 3a, the patients with reduced expression of PPAR b in tumor VECs (n = 120) exhibited a statistically favorable survival outcome, in contrast to those with increased PPAR b expression (n = 21) (P \ 0.001). By multivariate Cox regression analysis, the above significance remained independent of gender, age, location, stage, and differentiation [P \ 0.001
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J Gastroenterol Fig. 2 PPAR b expression in the vascular endothelial cells (VECs) of colorectal cancers and normal mucosa. A By immunohistochemical assay (IHC), PPAR b was predominantly detected in the cytoplasm of the VECs in colorectal cancers and the normal mucosa, with minor staining in the nuclei. All of the images were taken at a magnification of 9400. B The frequency of the cases with low expression (negative or weak staining) of PPAR b increased from the normal mucosa to the primary cancer samples (P \ 0.001)
(TNM stage), P = 0.003 (PPAR b expression)]. As shown in Table 2, the patients with reduced expression of PPAR b were 2.8 times less likely to die than those with elevated expression levels of PPAR b. In addition, we found that the patients in the T(high)V(low) group exhibited the best survival rate, whereas the cases in the T(low)V(high) group exhibited the worst prognoses (P = 0.002; Fig. 3b). The expression of PPAR b in the VECs of CRCs relative to the expression of VEGF, PINCH, CD31 and D2-40 We examined the expression of VEGF in the VECs of 92 CRCs. The frequency of the staining is presented in Fig. 4. VEGF localized in the cytoplasm and nucleus of the VECs within the tumor stroma. The elevated expression of VEGF in the VECs of CRCss (97.8 %, 90/92) was related to the reduced expression of PPAR b (81.5 %, 75/92, P \ 0.001). The data for PINCH (n = 80), CD31 (n = 116) and D240 (n = 116) of the primary CRCs was determined by IHC. As shown in Table 3, we found that reduced PPAR b expression was significantly correlated with increased D240 (P = 0.036) but not PINCH (P = 0.432) or CD31 (P = 0.323).
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Discussion Many studies have investigated the role of PPAR b in the pathogenesis of CRC, but the results are rather controversial. As the majority of studies have focused on the role of PPAR b in cancer cells, little is known about its role in the VECs of CRCs. To our knowledge, this is the first study to clarify this issue. We found that PPAR b predominantly localized within the cytoplasm of VECs, CRC cells and the epithelial cells of normal mucosa. The patients with low expression of PPAR b in the VECs of CRCs exhibited enhanced differentiation, earlier staging and favorable survival. The cases with increased expression of PPAR b in CRC cells with low expression in the VECs exhibited the best survival, and those with low expression of PPAR b in the CRC cells with high expression in the VECs exhibited the worst prognosis. The expression of PPAR b was negatively associated with the expression of VEGF and D2-40 in the VECs of CRCs. These findings indicate that the expression of PPAR b in VECs is negatively correlated with the development and the prognosis of CRC, which might be linked to the negative regulation of VEGF and D2-40 expression. PPAR b has been reported to play a pre-angiogenesis role in the VECs of lung and pancreatic cancer [9, 21],
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Fig. 3 The correlation between PPAR b expression in ECs of tumor stroma in primary colorectal cancers and survival. A Patients with low expression of PPAR b in the VECs of primary CRC exhibited more favorable disease-free survival, compared with the cases with increased expression (P \ 0.001). B Patients with a combination of low PPAR b expression levels in the VECs and high expression levels in the tumor cells of primary CRC exhibited more favorable diseasefree survival, compared with other combinatorial expression profiles (P = 0.002)
Table 2 Cox multivariate analysis and assessment of the prognostic significance of cytoplasmic PPAR b expression in primary tumor
§
The significance was analyzed by Cox regression model
whereas its role in the VECs of CRCs remains unclear. In this study, we found that the expression of PPAR b was significantly reduced in the VECs of CRCs compared to those of the normal mucosa. The CRC patients with low expression levels of PPAR b in the VECs exhibited enhanced differentiation, earlier staging and more favorable survival than those with relatively high expression levels of PPAR b. In contrast, no significant correlations of PPAR b expression in the VECs of CRC with gender, age, tumor location or growth pattern were found. These findings indicate that the expression of PPAR b in the VECs is negatively correlated with the development and the prognosis of CRC. Our previous study showed that the increased expression of PPAR b in rectal cancer cells was correlated with an increased frequency of stage I cases and a favorable prognosis for the rectal cancer patients [17]. In the present study, we combined the PPAR b expression in the tumor cells and in the VECs to analyze its correlation with the prognosis of CRC patients. Our results revealed that the increased expression levels of PPAR b in tumor cells but low expression levels in the VECs of CRCs were significantly correlated with the most favorable survival, while those with reduced expression levels of PPAR b in the tumor cells but elevated expression levels in the VECs exhibited the worst prognosis. These findings indicate that the role of PPAR b in the VECs might be opposite to its role in the CRC cells in terms of the development of CRC. The combination of PPAR b expression in the CRC cells and the VECs might represent an effective prognostic indicator for CRC. This finding appears to be quite interesting and confusing. A review by Muller suggests that PPAR b might elicit different functions or even divergent effects on tumor growth depending on whether it is localized in tumor cells or in tumor stroma [25], although the mechanism underlying this phenomenon remains unclear. Variant PPAR b isoforms, differential internal environment or signaling pathways in cancer cells and VECs might be attributable for these opposing roles.
Variables
Indicator of poor survival
HR (95 % CI)
P§
PPAR b expression
High vs. low
Low
2.8 (1.4–5.5)
Gender
Female vs. male
Male
0.8 (0.5–1.5)
0.509
Age
\70 vs. C70
C70
1.0 (1.0–1.1)
0.355
Location
Colon vs. rectum
Rectum
1.0 (0.5–1.8)
0.981
TNM stage
(I ? II) vs. (III ? IV)
III ? IV
5.4 (2.7–10.6)
Tumor grade
(Good ? moderate) vs. poor
Poor
2.0 (1.1–3.7)
0.003
\0.001 0.021
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To explore the mechanism underlying the correlation of PPAR b in the VECs with the TNM stage and the prognosis of the CRC patients, we further analyzed the correlation of PPAR b expression with that of VEGF, PINCH, CD31 and D2-40 in the CRCs. VEGF is the most prominent mediator of the angiogenic switch in malignant tumors [20], and PPAR b was recently shown to upregulate VEGF in several tumor types [26]. PINCH was shown to be overexpressed
Table 3 The correlation between PPAR b expression and PINCH, CD31, D2-40 expression in CRC patients Characteristics
Tumor stroma Positive (%)
P value Negative (%)
PINCH
0.432
Positive
4 (67)
37 (50)
Negative
2 (33)
37 (50)
Positive
6 (33)
45 (46)
Negative
12 (67)
53 (54)
CD31
0.323
D2-40
0.036
Positive
3 (17)
42 (43)
Negative
15 (83)
56 (57)
Fig. 4 The correlation between PPAR b expression in primary rectal cancers and VEGF expression. A By immunohistochemical assay (IHC), VEGF localized in the cytoplasm and nucleus of the VECs within the tumor stroma. The elevated expression of VEGF in the VECs of CRCs was related to the reduced expression of PPAR b. All of the images were taken at a magnification of 9400. B The patients demonstrated a negative correlation of PPAR b expression with the expression of VEGF (P \ 0.001)
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in the tumor-associated stroma of colorectal cancers and to be correlated with a less favorable clinical outcome [23, 27]. PINCH is involved in angiogenesis through the activation of fibroblasts in response to tumors [28, 29], indicating that PINCH is a likely regulator of angiogenesis. CD31 was identified to mediate the angiogenic and lymphangiogenic processes in the VECs of CRCs [24, 30, 31]. D2-40 is a cell surface sialylated glycoprotein and has been considered as a lymphatic-specific marker, which can stain lymphatic vessels in primary rectal cancer [32, 33]. In this study, we found that the reduced expression of PPAR b in the VECs of CRCs was significantly correlated with the increased expression of VEGF and D2-40 but not with PINCH and CD31. This finding indicates that PPAR b might also be involved in the angiogenesis of CRC via the direct or indirect regulation of VEGF and D2-40 but not with PINCH and CD31. Given that we have shown that the reduced expression of PPAR b in VECs was associated with favorable patient prognosis in this study, we thus hypothesize that the expression of PPAR b in VECs might promote the development and prognosis of CRC patients by regulating the angiogenesis of CRC that is mediated by VEGF and D2-40. However, the molecular mechanism for how PPAR b might play a role in the development of CRC requires further clarification in future studies.
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The specificity of the PPAR b antibody in this study was verified by immunofluorescence and Western blot analyses using three colon cancer cell lines of different stages. The two assays consistently showed that PPAR b predominantly localized in the cytoplasm and rarely in the nuclei of the cells, without the apparent differences among the cell lines of different stages. These data were consistent with the results of the predominantly cytoplasmic staining in the VECs, cancer cells and the epithelial cells of the normal mucosa, as determined by immunohistochemical analyses in this study. These findings strongly indicate that the PPAR b antibody used in this study was highly specific and that the expressed PPAR b predominantly localizes in the cytoplasm of VECs, CRC cells and of the epithelial cells of the normal mucosa. Consistently, Takayama et al. [34] and Yoshinaga et al. [35] also reported the predominant cytoplasmic expression of PPAR b in colon cancers. In contrast, PPAR b was initially reported to exhibit predominantly nuclear localization in colorectal epithelial cells, functioning as a nuclear receptor [36]. However, these studies did not further explore the expression pattern of PPAR b. This study is the first of its type to do so by Western blot of nuclear and cytoplasm protein fractions, as well as by immunofluorescence. Further studies are required to explain the cytoplasmic localization of PPAR b, in light of its known function as a nuclear receptor. In conclusion, PPAR is predominantly localized in the cytoplasm, and its expression is significantly decreased in the VECs of CRCs compared to that of the normal mucosa. The expression of PPAR is negatively correlated with the tumor differentiation, staging, and the survival of CRC patients, as well as the expression of VEGF and D2-40 in CRCs. These findings consistently indicate that PPAR b expression in the VECs might play a promotive role in the development of CRC, which might be linked to the angiogenesis mediated by VEGF and D2-40.
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14. Acknowledgments This study was supported by grants from the Natural Science Foundation of China (Grant No. 30801332), the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 200806100058), the Foundation for the Author of National Excellent Doctoral Dissertation of PR China (No. 2007B66), and the Swedish Cancer Foundation, Swedish Research Council and the Health Research Council in the South-East of Sweden. Conflict of interest Jin Zhou, Lie Yang, Yuan Li, Gunnar Arbman, Ke-Ling Chen, Bin Zhou, Yong-Yang Yu, Cun Wang, Xian-Ming Mo, You Lu, Zong-Guang Zhou and Xiao-Feng Sun declare no conflicts of interest relevant to this manuscript.
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