Med Oncol (2015) 32:414 DOI 10.1007/s12032-014-0414-9
ORIGINAL PAPER
Peroxiredoxin 2 is involved in vasculogenic mimicry formation by targeting VEGFR2 activation in colorectal cancer Shouru Zhang • Zhongxue Fu • Jinlai Wei Jinbao Guo • Maoxi Liu • Kunli Du
•
Received: 24 November 2014 / Accepted: 27 November 2014 / Published online: 4 December 2014 Ó Springer Science+Business Media New York 2014
Abstract The mammalian peroxiredoxin 2 (Prdx2) is a member of thiol-dependent antioxidant proteins and plays an important role in the progression of colorectal cancer (CRC). The aim of this study was to confirm the role of Prdx2 in formation of VM and progression of CRC. Immunohistochemistry and CD34/periodic acid Schiff double staining were performed to explore the expression of Prdx2 and VM formation in 70 CRC tissues, and there was a positive correlation between Prdx2 expression and VM formation by the Pearson correlation coefficient (r = 0.282, p \ 0.05). Prdx2 was suppressed in poorly differentiated HCT116 cells by Prdx2-siRNA-LV transduction. The expression of Prdx2 at both mRNA and protein levels in HCT116 cells transfected with the Prdx2 siRNA was significantly lower than that of negative control siRNA as confirmed by quantitative real-time PCR and Western blotting analysis, respectively (p \ 0.05). The well-established in vitro 3D culture model was chosen to investigate the VM formation of HCT116 cells. The numbers of the tubular structures were significantly fewer in Prdx2 siRNA explants than those of negative control siRNA explants after VEGF induction (p \ 0.05). Although VEGFR2 expressions had no change after VEGF induction, we found that VEGFR2 phosphorylation levels were markedly reduced in cells of siPrdx2 over time compared with those of negative control siRNA by Western blotting analysis (p \ 0.05, p \ 0.01). The effects of Prdx2 siRNA on the invasive capabilities of HCT116 cells with VEGF induction were examined by using Matrigel
S. Zhang Z. Fu (&) J. Wei J. Guo M. Liu K. Du Department of Gastrointestinal Surgery, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, People’s Republic of China e-mail: [email protected]
invasion chamber assay. The invasive capabilities of HCT116 cells were significantly declined in Prdx2 siRNA explants than those of negative control siRNA explants (p \ 0.05). The effects of Prdx2 siRNA on pathological tumor growth were examined by using a tumor xenograft model in vivo. After implant of HCT116 cells that transduced with Prdx2 siRNA and negative control siRNA as xenografts into nude mice, the growth of xenograft tumors with Prdx2 siRNA was much slower than that of negative control siRNA, and the volumes of tumor xenografts with Prdx2 siRNA were smaller than those of negative control siRNA after 5 weeks (p \ 0.05). Further conclusion showed that Prdx2 regulates VM formation by targeting VEGFR2 activation, which now represents as a therapeutic target for RC. Keywords Peroxiredoxin 2 Vasculogenic mimicry VEGFR2 Colorectal cancer
Introduction Colorectal cancer (CRC) is one of the most frequent human malignant neoplasms. Due to low physical activity, highcalorie and high-fat diet, obesity, and a sedentary lifestyle, the morbidity of CRC continues to rise. With the estimated incidence of more than 1,000,000 new cases annually worldwide, CRC is the third in malignant tumor of females and the second in males, respectively [1]. Although surgical resection and chemoradiotherapy have made great progress, CRC remains one of the leading causes of cancerrelated deaths worldwide [2]. Reactive oxygen species (ROS) that include the superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (OH-) are produced by cells as by-
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products of normal cellular metabolism. The elevated levels of ROS are well known for being both beneficial and deleterious [3]. Under normal physiological conditions, a balance is maintained between the production of ROS and the capacity of the antioxidant enzyme system. The recent understanding about ROS biology brought attention to the cellular functions of antioxidant enzymes. It is now well accepted that the peroxiredoxins (Prdxs) are the most important antioxidant enzymes [4]. The mammalian Prdxs are in a superfamily of thiol-dependent antioxidant proteins that have six members (Prdx1–6). The elevated expression levels of individual Prdx isoforms have been detected in several human carcinomas [5, 6]. Among them, Prdx2 is upregulated in CRC and contributes to CRC cells’ survival by protecting cells from oxidative stress [7]. Prdx2 knockdown inhibits the growth of CRC cells [8]. In the absence of Prdx2, the cellular H2O2 level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation in endothelial cells. Prdx2 knockdown suppresses tumor angiogenesis in vivo [9]. A novel phenomenon of blood vessel formation by melanoma cells was reported and termed as vasculogenic mimicry (VM) [10]. VM describes the functional plasticity of aggressive cancer cells forming vascular networks, thereby providing a perfusion pathway for rapidly growing tumors. The presence of VM in tumor tissues has been associated with a poor clinical outcome. In recent years, the presence of VM has been detected in several human carcinomas, such as CRC [11]. Although the molecular mechanism of VM channel formation is still not clear, VEGF plays a crucial role in the formation of VM [12, 13], and VEGF receptor tyrosine kinases such as VEGFR2 (also known as KDR or Flk-1) bind VEGF in an autocrine or paracrine manner and demonstrate many signaling capacities in the formation of VM channels [14, 15]. Since it was proved that Prdx2 is upregulated in CRC, we suspected that Prdx2 plays a significant role in the formation of VM channels. In the present study, we found that Prdx2 expressions were associated with VM formation in CRC tissues and knockdown of Prdx2 inhibited VEGFinduced VM formation of CRC cells by targeting VEGFR2 activation.
Materials and methods Patients and tissue samples The study consisted of 70 CRC patients who had not undergone chemotherapy or radiotherapy prior to surgery between March 2012 and June 2012. The colorectal specimens were obtained from the Department of Gastrointestinal Surgery, the First Affiliated Hospital of Chongqing
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Medical University. The histological type of the specimens was reviewed independently by two senior pathologists. The use of the tissue samples was approved by the Medical Ethics Review Committee of the First Affiliated Hospital of Chongqing Medical University. Immunohistochemistry and CD34/periodic acid Schiff double staining. The immunohistochemistry (IHC) staining was performed using an immunohistochemical SP-9000 kit (Zhongshan Chemical, Beijing, China). Formalin-fixed and paraffinembedded sections were deparaffinized, and then, antigens were retrieved by heating in a microwave oven at 90 °C for 10 min in citrate buffer. The sections were then incubated in 3 % hydrogen peroxide for 15 min and blocked with goat serum albumin for 30 min at room temperature. After incubation with rabbit anti-Prdx2 (1:100) primary antibodies overnight at 4 °C, the sections washed with PBS the next day, the secondary antibodies were applied for 30 min at room temperature and then washed with PBS. After incubated with streptavidin-HRP for 30 min at room temperature and rinsed with PBS, the sections were incubated for 15 min with the chromogen 3,3-diaminobenzidine and counterstained with hematoxylin. Finally, after dehydrated and mounted, the sections were examined under the transmission light microscope. The CD34/periodic acid Schiff double staining was performed according to the manufacturer’s instructions (Senbeijia Chemical, Nanjing, China). After immunohistochemical (IHC) staining for CD34 (Zhongshan Chemical, Beijing, China) as above, the sections were washed with running water for 6 min, incubated with periodic acid Schiff for 15 min and counterstained with hematoxylin. Finally, after dehydrated and mounted, the sections were examined under the transmission light microscope. Reagents and antibodies Recombinant human VEGF165 was purchased from PeproTech (Rocky Hill, NJ, USA). The following antibodies were purchased for use: rabbit anti-Prdx2 (Abcam, San Francisco, CA, USA); rabbit anti-CD34 (Zhongshan Chemical, Beijing, China); rabbit anti-VEGFR2 (Biosynthesis, Beijing, China); rabbit anti-phospho-VEGF receptor 2(Tyr1175) (CST, Danvers, MA, USA); and mouse antiGAPDH (Beyotime, Jiangsu, China). Cell culture and 3D culture Human CRC cell line HCT116 was purchased from the Shanghai Cell Bank at the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Leibovitz
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L-15 medium (Gibco, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (FBS) (TBD, Tianjin, China) and 2 % penicillin/streptomycin (Beyotime, Jiangsu, China), and they were maintained at 37 °C and 5 % CO2. For VM formation, human CRC cell line HCT116 was cultured for 18 h in Leibovitz L-15 serumfree culture medium for the assay and then placed on 12-well culture dishes (5 9 105cells/well) containing Matrigel matrix (BD Bioscience, NJ, USA) in the presence of VEGF165 (50 ng/ml). After incubation at 37 °C for 48 h, the numbers of tubelike channels were measured from five random visual fields per sample. Transfection analysis using siRNA The Prdx2 siRNA vector sequences (forward 50 -TCC TCT TTA TCA TCG ATG GCA ACT CGA GTT GCC ATC GAT GAT AAA GAG GTT TTT TC-30 and reverse 30 -TCG AGA AAA AAC CTC TTT ATC ATC GAT GGC AAC TCG AGT TGC CAT CGA TGA TAA AGA GGA-50 ) were used to downregulate Prdx2 according to the instructions (Genechem, Shanghai, China). HCT116 cells were seeded in 6-well plates at a concentration of 0.5 9 105 per well (20–30 % confluence) on the day before transfection. Prdx2-siRNA-LV was transduced into cells at an MOI of 40 by using polybrene (10 lg/ml) and enhanced infection solution (Genechem, Shanghai, China). A non-target negative control virus GFP-LV (Genechem, Shanghai, China) was transduced into cells used as a negative control. The enhanced infection solution was replaced with L-15 medium supplemented with 10 % fetal bovine serum after incubation for 12 h. The validity of transfection was detected 72 h after transfection. Quantitative RT-PCR analysis Total RNA was extracted from cells using Trizol Reagent (TaKaRa, Dalian, China) as recommended by the manufacturer. The concentration and purity of RNA were quantified using a UV spectrophotometer (UltroSPec2100 Pro, Amersham, USA). Total RNA was reverse transcribed using Prime Script RT Reagent kit TaKaRa, Dalian, China) in a total volume of 20 ll according to the manufacturer’s instructions. Quantitative PCR (qRT-PCR) was performed by using SYBR Premix Ex Taq II (TaKaRa, Dalian, China). Primer sequences used for the amplification of human genes were as follows: Prdx2 (forward 50 -CAC CTG GCT TGG ATC AAC ACC-30 and reverse 50 -CAG CAC GCC GTA ATC CTC AG-30 ) and GAPDH (forward 50 -ACC ACA GTC CAT GCC ATC CAC-3 and reverse 50 -TCC ACC ACC CTG TTG CTG TA-30 ). GAPDH was used as an internal control. The relative expression levels
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of mRNAs were quantified using the 2 ðD Ct sample D Ct controlÞ method. Each experiment was performed in triplicate. Western blotting analysis The cells at exponential stage were harvested and then lysed in lyses buffer (50 mM Tris, 150 mM NaCl, 1 % NP40, 0.1 % SDS, 10 mM EDTA, 1 mM PMSF, and 0.5 % sodium deoxycholate) according to the instructions (KeyGEN, Nanjing, China). After centrifuging at 12,000 rpm at 4 °C for 15 min, the supernatants of lysates were collected for use. The proteins concentration was quantified by using the BCA protein assay kit (Beyotime, Jiangsu, China). The indicated amounts of proteins were separated by SDSPAGE and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5 % BSA for 2 h and then incubated with primary antibodies overnight at 4 °C: rabbit anti-Prdx2 (1:1,000); rabbit anti-VEGFR2 (1:500); rabbit anti-Phospho VEGF receptor 2 (Tyr1175) (1:1,000); and mouse anti-GAPDH (1:300). After washing with PBST 15 min, the membranes were incubated with secondary antibodies (1:5,000) at 37 °C for 1 h. After washing with PBST 15 min, the detection was performed using an enhanced chemiluminescence kit (KeyGEN, Nanjing, China). Specific bands were quantified using Quantity One 4.6.2. Each experiment was performed in triplicate. Matrigel invasion chamber assay HCT116 cells that transduced with Prdx2 siRNA and negative control were cultured in Leibovitz L-15 serumfree culture medium at 37 °C with 5 % CO2 for 18 h before the assay. Matrigel matrix (BD Bioscience, NJ, USA) with a final concentration of 1.5 mg/ml was added to the upper surface of each transwell chamber filter with 8-lm membrane pores (Millipore, Bedford, MA, USA) and incubated at 37 °C with 5 % CO2 for 30 min. The serumfree culture medium at 300 ll was added to the lower chamber supplemented with VEGF165 (50 ng/ml), and the trypsinized cells at 200 ll (5 9 105cells/ml) contained in a serum-free culture medium was added to the upper chamber. After incubation at 37 °C with 5 % CO2 for 40 h, the upper chamber was cleaned with PBS. The invading cells were fixed with 4 % formaldehyde for 30 min and stained with 0.5 % crystal violet. The numbers of invading cells were quantified under the transmission light microscope. Animal studies Five-week-old male BALB/c nude mice were purchased from the Laboratory Animal Center of Chongqing Medical
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University. The mice were raised in a specific pathogenfree unit under isothermal conditions. All experimental procedures were complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. HCT116 cells (3 9 106) that transduced with Prdx2 siRNA or negative control vector were suspended in 100 ll of serum-free medium and then implanted subcutaneously into the right flank of nude mice. Tumor diameters were measured every week using vernier calipers when the tumor size reached approximately 100 mm3. The tumor volumes were calculated using the following formula: vðmm3 Þ ¼ length width2 =2: The tumors were harvested for the analysis after 5 weeks. Statistical analysis SPSS 18.0 (SPSS, Inc, Chicago, IL, USA) was used for statistical analyses. p \ 0.05 was considered statistically significant. Differences in quantitative data between groups were analyzed using the Student’s t test, and enumeration data were analyzed using the Chi-squared test. The associations between the expression levels of Prdx2 and VM were analyzed by the Pearson correlation coefficient. The data for invasion and VM formation assays were analyzed using one-way ANOVA test.
Fig. 1 Immunohistochemical staining of Prdx2 in colorectal carcinoma and normal tissues, CD34/PAS double staining of VM in colorectal carcinoma tissues. a Strong staining of Prdx2 is observed in colorectal carcinoma tissues (9200). b Weak staining of Prdx2 is observed in normal colorectal mucosa tissues (9200). c The vasculogenic mimicry channels lined with tumor cells (red arrows, CD34/ PAS double staining, 9400). d The endothelial-dependent vessels are both positive for CD34 and PAS (black arrows, CD34/PAS double staining, 9400)
Results
Table 1 Correlation of Prdx2 and VM expression in CRC tissue samples
Expression of Prdx2 and vasculogenic mimicry (VM) in CRC tissues
VM
The expressions of Prdx2 were examined in CRC tissues by using IHC. Prdx2 expression was predominantly located in the cytoplasm and nucleus of CRC cells (Fig. 1a). The positive staining of Prdx2 in CRC tissues was 70.00 % (49/ 70). There is no staining or only weak staining was observed in normal colorectal mucosa tissues (p \ 0.05, Fig. 1b). VM was detected in CRC tissues by using CD34/ periodic acid Schiff (PAS) double staining. VM channels were composed of CRC cells negative for CD34 (pointed out by the red arrows in Fig. 1c), but the endothelialdependent vessels were both positive for CD34 and PAS (pointed out by the black arrows in Fig. 1d). All the base membrane-like structures between red blood cells and tumor cells were positive for PAS. The positive staining of VM in CRC tissues was 22.86 % (16/70). There was a positive correlation between Prdx2 expression and VM formation by the Pearson correlation coefficient (r = 0.282, p \ 0.05). The correlation between Prdx2 expression and VM formation is shown in Table 1. The
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Prdx2 Positive (n = 49)
Negative (n = 21)
Positive (n = 16)
15
1
Negative (n = 54)
34
20
Pearson correlation
p value
0.282
0.018
results indicate that Prdx2 expression is associated with VM formation in CRC. Knockdown of Prdx2 inhibits VEGF-induced VM formation of HCT116 cells and impairs VEGFR2 activation The poorly differentiated HCT116 cell was chosen to investigate the VM formation by using the well-established in vitro 3D culture model. So Prdx2 was suppressed in HCT116 cells by Prdx2-siRNA-LV transduction. The expression of Prdx2 at both mRNA and protein levels in HCT116 cells transfected with the Prdx2 siRNA was significantly lower than that of negative
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control siRNA, as confirmed by quantitative real-time PCR and Western blotting analysis, respectively (p \ 0.05, Fig. 2a–c). VEGF-VEGFR2 axis has a critical role in VM formation, VEGF plays a crucial role in the formation of VM in an autocrine or paracrine manner, so recombinant human VEGF induction was used to mimic the microenvironment in vitro. We found that the numbers of the tubular structures were significantly decreased in Prdx2 siRNA explants than those of negative control siRNA explants after inducing by VEGF (p \ 0.01, Fig. 3a–c). The result indicates that Prdx2 knockdown impedes the VEGF-induced VM formation. The expressions of VEGFR2 and phospho-VEGFR2 were assayed by Western blotting in HCT116 cells transduced with Prdx2 siRNA and negative control siRNA after induced by VEGF at different time periods of culture. Although VEGFR2 protein expressions had no change, we found that VEGFR2 phosphorylation levels induced by VEGF were markedly reduced in cells transduced with Prdx2 siRNA compared with those of negative control siRNA by Western blotting analysis over time (p \ 0.05, p \ 0.01, Fig. 3d, e). These data indicate that Prdx2 knockdown inhibits VEGF-induced VM formation by reducing the phosphorylation level of VEGFR2. Knockdown of Prdx2 inhibits VEGF-induced invasion in HCT116 cell in vitro The effects of Prdx2 knockdown on the invasive capabilities of HCT116 cells with VEGF induction were examined by Matrigel invasion chamber assay. After VEGF induction, the invasive capabilities of HCT116 cells were significantly decreased in Prdx2 siRNA explants than those of negative control siRNA explants (p \ 0.05, Fig. 4). These data indicate that Prdx2 is a critical element of invasion in CRC progression and plays an important role in the progression of CRC.
Fig. 2 Prdx2 expression is suppressed by Prdx2 siRNA in HCT116cells. a Prdx2 mRNA expression in HCT116 cells transfected with Prdx2 siRNA is significantly reduced than that of negative control siRNA, as determined by qRT-PCR analysis (p \ 0.05). b,
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Knockdown of Prdx2 inhibits tumor growth in vivo Lastly, we examined the effect of Prdx2 deficiency on pathological tumor growth using a tumor xenograft model in vivo. After implant of HCT116 cell lines that transduced with Prdx2 siRNA and negative control siRNA as xenografts into nude mice, the growth of xenograft tumors with Prdx2 siRNA was much slower than that of negative control siRNA. After growing for 5 weeks, the volumes of tumor xenografts with Prdx2 siRNA were smaller than those of negative control siRNA (p \ 0.05, Fig. 5). These data indicate that Prdx2 Knockdown can retard tumor growth in CRC.
Discussion CRC is still the main tumor threatening to people’s health, it is vital to explore new molecular pathways as its incidence increases year by year [16]. Our team had previously reported that Prdx2 expression was elevated in CRC tissues and was significantly associated with tumor metastasis and the TNM stage (7). Prdx2 expression was closely related to the low degree of tumor differentiation and status of lymph node metastasis (22). Here, we detected Prdx2 expression in more CRC tissues than before and confirmed that Prdx2 overexpression existed in CRC tissues. To the best of our knowledge, it had been proved that VM-positive tumor patients also had more risks of metastasis and recurrence, and VM-positive expression was closely related to the low degree of tumor differentiation (21). Since Maniotis first confirmed the existence of VM in uveal melanoma, VM had been identified successively in many tumors, including gastric stromal tumor [17], ovarian cancer [12], prostate cancer [18], liver cancer [19], breast cancer [20], and CRC [11]. Prdx2 expression and VM-positive expression are both closely related to low degree of tumor differentiation
c Prdx2 protein expression is suppressed in cells after transfection with Prdx2 siRNA compared with that of negative control siRNA, as shown by Western blotting analysis (p \ 0.05)
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Fig. 3 Effect of Prdx2 on the vasculogenic mimicry formation of HCT 116 cell in 3D culture. a–c After induced by VEGF165 (50 ng/ ml), VM channels are significantly reduced in siPrdx2 explants than those of negative control siRNA explants (9200, p \ 0.01). d, e After
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induced by VEGF165 (10 ng/ml), VEGFR2 expression had no change, phospho-VEGFR2 is markedly reduced over time in cells of Prdx2 siRNA compared with that of negative control siRNA, as by Western blotting analysis (p \ 0.05, p \ 0.01)
Fig. 4 Effect of siPrdx2 on the invasive capabilities of HCT 116 cells. a–c After induced by VEGF165 (50 ng/ml), the invasive capabilities of HCT116 cells are significantly decreased in Prdx2 siRNA explants than those of negative control siRNA explants (p \ 0.05, 9200)
Fig. 5 Prdx2 knockdown inhibited the growth of xenograft tumors in nude mice. a Representative xenograft tumors are shown. b Tumor volumes are monitored over time (*p \ 0.05)
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and adverse clinical outcome, and we demonstrated a critical role of Prdx2 in the formation of VM. In our study, the incidence of VM in CRC tissues was 22.86 % (16/70), which was similar to that previously reported [21]. We found that the overexpression of Prdx2 was associated with the incidence of VM in CRC tissues. Our study also provided the evidence that the poorly differentiated CRC HCT116 cells can form obvious VM channels in vitro by using a well-established 3D culture model which was similar to that previously reported [21]. Unlike tubular channels formed by HUVEC cells, the tubular channels of HCT116 cells were made up of several layers of cells which were thicker and denser than those of HUVEC cells. As a selective cytokine, VEGF can secrete with the microenvironment changes and plays a crucial role in the formation of VM [22]. VEGFR2, as the major VEGF receptor requires phosphorylation for activation, demonstrates many signaling capacities in the formation of VM channels [23–25]. It was reported that VEGFR2 was expressed in tumor cells from patients with CRC and responded to VEGF stimulation with augmented VEGFR2-mediated proliferation and tumor growth, and the activation of VEGF receptors on tumor cells could mediate tumor growth and metastasis [26]. It has been shown that Prdxs were lost the peroxidase activity by inhibitory phosphorylation on threonine or tyrosine residue [27]. Our study showed that VEGFR2 expression was not changed significantly after induced by VEGF, but VEGFR2 phosphorylation levels were much lower over time in CRC cells of Prdx2 siRNA compared with those of negative control cells. We confirmed that Prdx2 is a key stimulatory molecule in VM formation by involving in maintenance of VEGFR2 activation. Invasion and metastasis are important characters of malignant tumors and main obstacles to cure, the invasive capabilities of cancer cells are closely related to VM formation and adverse clinical outcome, and targeting the vasculature remains a promising approach for treating solid tumors [28, 29]. The effect of Prdx2 knockdown on the invasive capabilities of CRC cells with VEGF induction was examined. After VEGF induction, the invasive capabilities of CRC cells were significantly decreased in Prdx2 knockdown explants than those of negative control siRNA explants. The effect of Prdx2 deficiency on pathological tumor growth using a tumor xenograft model in vivo also showed that Prdx2 was critical for pathological tumor growth. The tumor growth in Prdx2 deficiency mice was much slower than that of negative control siRNA mice. As a special mode of blood supply in addition to angiogenesis, VM is closely related to tumor cell growth activity. The growth and metastasis of cancer can be retarded by interfering with the formation of VM vessels [30]. We think that Prdx2 deficiency can retard CRC cells growth partly due to reduced VM vessels in vivo, but further research of mechanism is needed.
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In summary, one of our important findings is that as a major antioxidant enzyme, Prdx2 mediates VM formation by regulating VEGFR2 activation, which now represents as a therapeutic target for CRC. Acknowledgments This study was supported by a Grant from the National Natural Science Foundation of China (No. 81172295). Conflict of interest interest.
All the authors indicate no potential conflict of
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ORIGINAL PAPER
Peroxiredoxin 2 is involved in vasculogenic mimicry formation by targeting VEGFR2 activation in colorectal cancer Shouru Zhang • Zhongxue Fu • Jinlai Wei Jinbao Guo • Maoxi Liu • Kunli Du
•
Received: 24 November 2014 / Accepted: 27 November 2014 / Published online: 4 December 2014 Ó Springer Science+Business Media New York 2014
Abstract The mammalian peroxiredoxin 2 (Prdx2) is a member of thiol-dependent antioxidant proteins and plays an important role in the progression of colorectal cancer (CRC). The aim of this study was to confirm the role of Prdx2 in formation of VM and progression of CRC. Immunohistochemistry and CD34/periodic acid Schiff double staining were performed to explore the expression of Prdx2 and VM formation in 70 CRC tissues, and there was a positive correlation between Prdx2 expression and VM formation by the Pearson correlation coefficient (r = 0.282, p \ 0.05). Prdx2 was suppressed in poorly differentiated HCT116 cells by Prdx2-siRNA-LV transduction. The expression of Prdx2 at both mRNA and protein levels in HCT116 cells transfected with the Prdx2 siRNA was significantly lower than that of negative control siRNA as confirmed by quantitative real-time PCR and Western blotting analysis, respectively (p \ 0.05). The well-established in vitro 3D culture model was chosen to investigate the VM formation of HCT116 cells. The numbers of the tubular structures were significantly fewer in Prdx2 siRNA explants than those of negative control siRNA explants after VEGF induction (p \ 0.05). Although VEGFR2 expressions had no change after VEGF induction, we found that VEGFR2 phosphorylation levels were markedly reduced in cells of siPrdx2 over time compared with those of negative control siRNA by Western blotting analysis (p \ 0.05, p \ 0.01). The effects of Prdx2 siRNA on the invasive capabilities of HCT116 cells with VEGF induction were examined by using Matrigel
S. Zhang Z. Fu (&) J. Wei J. Guo M. Liu K. Du Department of Gastrointestinal Surgery, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, People’s Republic of China e-mail: [email protected]
invasion chamber assay. The invasive capabilities of HCT116 cells were significantly declined in Prdx2 siRNA explants than those of negative control siRNA explants (p \ 0.05). The effects of Prdx2 siRNA on pathological tumor growth were examined by using a tumor xenograft model in vivo. After implant of HCT116 cells that transduced with Prdx2 siRNA and negative control siRNA as xenografts into nude mice, the growth of xenograft tumors with Prdx2 siRNA was much slower than that of negative control siRNA, and the volumes of tumor xenografts with Prdx2 siRNA were smaller than those of negative control siRNA after 5 weeks (p \ 0.05). Further conclusion showed that Prdx2 regulates VM formation by targeting VEGFR2 activation, which now represents as a therapeutic target for RC. Keywords Peroxiredoxin 2 Vasculogenic mimicry VEGFR2 Colorectal cancer
Introduction Colorectal cancer (CRC) is one of the most frequent human malignant neoplasms. Due to low physical activity, highcalorie and high-fat diet, obesity, and a sedentary lifestyle, the morbidity of CRC continues to rise. With the estimated incidence of more than 1,000,000 new cases annually worldwide, CRC is the third in malignant tumor of females and the second in males, respectively [1]. Although surgical resection and chemoradiotherapy have made great progress, CRC remains one of the leading causes of cancerrelated deaths worldwide [2]. Reactive oxygen species (ROS) that include the superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radicals (OH-) are produced by cells as by-
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products of normal cellular metabolism. The elevated levels of ROS are well known for being both beneficial and deleterious [3]. Under normal physiological conditions, a balance is maintained between the production of ROS and the capacity of the antioxidant enzyme system. The recent understanding about ROS biology brought attention to the cellular functions of antioxidant enzymes. It is now well accepted that the peroxiredoxins (Prdxs) are the most important antioxidant enzymes [4]. The mammalian Prdxs are in a superfamily of thiol-dependent antioxidant proteins that have six members (Prdx1–6). The elevated expression levels of individual Prdx isoforms have been detected in several human carcinomas [5, 6]. Among them, Prdx2 is upregulated in CRC and contributes to CRC cells’ survival by protecting cells from oxidative stress [7]. Prdx2 knockdown inhibits the growth of CRC cells [8]. In the absence of Prdx2, the cellular H2O2 level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation in endothelial cells. Prdx2 knockdown suppresses tumor angiogenesis in vivo [9]. A novel phenomenon of blood vessel formation by melanoma cells was reported and termed as vasculogenic mimicry (VM) [10]. VM describes the functional plasticity of aggressive cancer cells forming vascular networks, thereby providing a perfusion pathway for rapidly growing tumors. The presence of VM in tumor tissues has been associated with a poor clinical outcome. In recent years, the presence of VM has been detected in several human carcinomas, such as CRC [11]. Although the molecular mechanism of VM channel formation is still not clear, VEGF plays a crucial role in the formation of VM [12, 13], and VEGF receptor tyrosine kinases such as VEGFR2 (also known as KDR or Flk-1) bind VEGF in an autocrine or paracrine manner and demonstrate many signaling capacities in the formation of VM channels [14, 15]. Since it was proved that Prdx2 is upregulated in CRC, we suspected that Prdx2 plays a significant role in the formation of VM channels. In the present study, we found that Prdx2 expressions were associated with VM formation in CRC tissues and knockdown of Prdx2 inhibited VEGFinduced VM formation of CRC cells by targeting VEGFR2 activation.
Materials and methods Patients and tissue samples The study consisted of 70 CRC patients who had not undergone chemotherapy or radiotherapy prior to surgery between March 2012 and June 2012. The colorectal specimens were obtained from the Department of Gastrointestinal Surgery, the First Affiliated Hospital of Chongqing
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Medical University. The histological type of the specimens was reviewed independently by two senior pathologists. The use of the tissue samples was approved by the Medical Ethics Review Committee of the First Affiliated Hospital of Chongqing Medical University. Immunohistochemistry and CD34/periodic acid Schiff double staining. The immunohistochemistry (IHC) staining was performed using an immunohistochemical SP-9000 kit (Zhongshan Chemical, Beijing, China). Formalin-fixed and paraffinembedded sections were deparaffinized, and then, antigens were retrieved by heating in a microwave oven at 90 °C for 10 min in citrate buffer. The sections were then incubated in 3 % hydrogen peroxide for 15 min and blocked with goat serum albumin for 30 min at room temperature. After incubation with rabbit anti-Prdx2 (1:100) primary antibodies overnight at 4 °C, the sections washed with PBS the next day, the secondary antibodies were applied for 30 min at room temperature and then washed with PBS. After incubated with streptavidin-HRP for 30 min at room temperature and rinsed with PBS, the sections were incubated for 15 min with the chromogen 3,3-diaminobenzidine and counterstained with hematoxylin. Finally, after dehydrated and mounted, the sections were examined under the transmission light microscope. The CD34/periodic acid Schiff double staining was performed according to the manufacturer’s instructions (Senbeijia Chemical, Nanjing, China). After immunohistochemical (IHC) staining for CD34 (Zhongshan Chemical, Beijing, China) as above, the sections were washed with running water for 6 min, incubated with periodic acid Schiff for 15 min and counterstained with hematoxylin. Finally, after dehydrated and mounted, the sections were examined under the transmission light microscope. Reagents and antibodies Recombinant human VEGF165 was purchased from PeproTech (Rocky Hill, NJ, USA). The following antibodies were purchased for use: rabbit anti-Prdx2 (Abcam, San Francisco, CA, USA); rabbit anti-CD34 (Zhongshan Chemical, Beijing, China); rabbit anti-VEGFR2 (Biosynthesis, Beijing, China); rabbit anti-phospho-VEGF receptor 2(Tyr1175) (CST, Danvers, MA, USA); and mouse antiGAPDH (Beyotime, Jiangsu, China). Cell culture and 3D culture Human CRC cell line HCT116 was purchased from the Shanghai Cell Bank at the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Leibovitz
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L-15 medium (Gibco, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (FBS) (TBD, Tianjin, China) and 2 % penicillin/streptomycin (Beyotime, Jiangsu, China), and they were maintained at 37 °C and 5 % CO2. For VM formation, human CRC cell line HCT116 was cultured for 18 h in Leibovitz L-15 serumfree culture medium for the assay and then placed on 12-well culture dishes (5 9 105cells/well) containing Matrigel matrix (BD Bioscience, NJ, USA) in the presence of VEGF165 (50 ng/ml). After incubation at 37 °C for 48 h, the numbers of tubelike channels were measured from five random visual fields per sample. Transfection analysis using siRNA The Prdx2 siRNA vector sequences (forward 50 -TCC TCT TTA TCA TCG ATG GCA ACT CGA GTT GCC ATC GAT GAT AAA GAG GTT TTT TC-30 and reverse 30 -TCG AGA AAA AAC CTC TTT ATC ATC GAT GGC AAC TCG AGT TGC CAT CGA TGA TAA AGA GGA-50 ) were used to downregulate Prdx2 according to the instructions (Genechem, Shanghai, China). HCT116 cells were seeded in 6-well plates at a concentration of 0.5 9 105 per well (20–30 % confluence) on the day before transfection. Prdx2-siRNA-LV was transduced into cells at an MOI of 40 by using polybrene (10 lg/ml) and enhanced infection solution (Genechem, Shanghai, China). A non-target negative control virus GFP-LV (Genechem, Shanghai, China) was transduced into cells used as a negative control. The enhanced infection solution was replaced with L-15 medium supplemented with 10 % fetal bovine serum after incubation for 12 h. The validity of transfection was detected 72 h after transfection. Quantitative RT-PCR analysis Total RNA was extracted from cells using Trizol Reagent (TaKaRa, Dalian, China) as recommended by the manufacturer. The concentration and purity of RNA were quantified using a UV spectrophotometer (UltroSPec2100 Pro, Amersham, USA). Total RNA was reverse transcribed using Prime Script RT Reagent kit TaKaRa, Dalian, China) in a total volume of 20 ll according to the manufacturer’s instructions. Quantitative PCR (qRT-PCR) was performed by using SYBR Premix Ex Taq II (TaKaRa, Dalian, China). Primer sequences used for the amplification of human genes were as follows: Prdx2 (forward 50 -CAC CTG GCT TGG ATC AAC ACC-30 and reverse 50 -CAG CAC GCC GTA ATC CTC AG-30 ) and GAPDH (forward 50 -ACC ACA GTC CAT GCC ATC CAC-3 and reverse 50 -TCC ACC ACC CTG TTG CTG TA-30 ). GAPDH was used as an internal control. The relative expression levels
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of mRNAs were quantified using the 2 ðD Ct sample D Ct controlÞ method. Each experiment was performed in triplicate. Western blotting analysis The cells at exponential stage were harvested and then lysed in lyses buffer (50 mM Tris, 150 mM NaCl, 1 % NP40, 0.1 % SDS, 10 mM EDTA, 1 mM PMSF, and 0.5 % sodium deoxycholate) according to the instructions (KeyGEN, Nanjing, China). After centrifuging at 12,000 rpm at 4 °C for 15 min, the supernatants of lysates were collected for use. The proteins concentration was quantified by using the BCA protein assay kit (Beyotime, Jiangsu, China). The indicated amounts of proteins were separated by SDSPAGE and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5 % BSA for 2 h and then incubated with primary antibodies overnight at 4 °C: rabbit anti-Prdx2 (1:1,000); rabbit anti-VEGFR2 (1:500); rabbit anti-Phospho VEGF receptor 2 (Tyr1175) (1:1,000); and mouse anti-GAPDH (1:300). After washing with PBST 15 min, the membranes were incubated with secondary antibodies (1:5,000) at 37 °C for 1 h. After washing with PBST 15 min, the detection was performed using an enhanced chemiluminescence kit (KeyGEN, Nanjing, China). Specific bands were quantified using Quantity One 4.6.2. Each experiment was performed in triplicate. Matrigel invasion chamber assay HCT116 cells that transduced with Prdx2 siRNA and negative control were cultured in Leibovitz L-15 serumfree culture medium at 37 °C with 5 % CO2 for 18 h before the assay. Matrigel matrix (BD Bioscience, NJ, USA) with a final concentration of 1.5 mg/ml was added to the upper surface of each transwell chamber filter with 8-lm membrane pores (Millipore, Bedford, MA, USA) and incubated at 37 °C with 5 % CO2 for 30 min. The serumfree culture medium at 300 ll was added to the lower chamber supplemented with VEGF165 (50 ng/ml), and the trypsinized cells at 200 ll (5 9 105cells/ml) contained in a serum-free culture medium was added to the upper chamber. After incubation at 37 °C with 5 % CO2 for 40 h, the upper chamber was cleaned with PBS. The invading cells were fixed with 4 % formaldehyde for 30 min and stained with 0.5 % crystal violet. The numbers of invading cells were quantified under the transmission light microscope. Animal studies Five-week-old male BALB/c nude mice were purchased from the Laboratory Animal Center of Chongqing Medical
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University. The mice were raised in a specific pathogenfree unit under isothermal conditions. All experimental procedures were complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. HCT116 cells (3 9 106) that transduced with Prdx2 siRNA or negative control vector were suspended in 100 ll of serum-free medium and then implanted subcutaneously into the right flank of nude mice. Tumor diameters were measured every week using vernier calipers when the tumor size reached approximately 100 mm3. The tumor volumes were calculated using the following formula: vðmm3 Þ ¼ length width2 =2: The tumors were harvested for the analysis after 5 weeks. Statistical analysis SPSS 18.0 (SPSS, Inc, Chicago, IL, USA) was used for statistical analyses. p \ 0.05 was considered statistically significant. Differences in quantitative data between groups were analyzed using the Student’s t test, and enumeration data were analyzed using the Chi-squared test. The associations between the expression levels of Prdx2 and VM were analyzed by the Pearson correlation coefficient. The data for invasion and VM formation assays were analyzed using one-way ANOVA test.
Fig. 1 Immunohistochemical staining of Prdx2 in colorectal carcinoma and normal tissues, CD34/PAS double staining of VM in colorectal carcinoma tissues. a Strong staining of Prdx2 is observed in colorectal carcinoma tissues (9200). b Weak staining of Prdx2 is observed in normal colorectal mucosa tissues (9200). c The vasculogenic mimicry channels lined with tumor cells (red arrows, CD34/ PAS double staining, 9400). d The endothelial-dependent vessels are both positive for CD34 and PAS (black arrows, CD34/PAS double staining, 9400)
Results
Table 1 Correlation of Prdx2 and VM expression in CRC tissue samples
Expression of Prdx2 and vasculogenic mimicry (VM) in CRC tissues
VM
The expressions of Prdx2 were examined in CRC tissues by using IHC. Prdx2 expression was predominantly located in the cytoplasm and nucleus of CRC cells (Fig. 1a). The positive staining of Prdx2 in CRC tissues was 70.00 % (49/ 70). There is no staining or only weak staining was observed in normal colorectal mucosa tissues (p \ 0.05, Fig. 1b). VM was detected in CRC tissues by using CD34/ periodic acid Schiff (PAS) double staining. VM channels were composed of CRC cells negative for CD34 (pointed out by the red arrows in Fig. 1c), but the endothelialdependent vessels were both positive for CD34 and PAS (pointed out by the black arrows in Fig. 1d). All the base membrane-like structures between red blood cells and tumor cells were positive for PAS. The positive staining of VM in CRC tissues was 22.86 % (16/70). There was a positive correlation between Prdx2 expression and VM formation by the Pearson correlation coefficient (r = 0.282, p \ 0.05). The correlation between Prdx2 expression and VM formation is shown in Table 1. The
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Prdx2 Positive (n = 49)
Negative (n = 21)
Positive (n = 16)
15
1
Negative (n = 54)
34
20
Pearson correlation
p value
0.282
0.018
results indicate that Prdx2 expression is associated with VM formation in CRC. Knockdown of Prdx2 inhibits VEGF-induced VM formation of HCT116 cells and impairs VEGFR2 activation The poorly differentiated HCT116 cell was chosen to investigate the VM formation by using the well-established in vitro 3D culture model. So Prdx2 was suppressed in HCT116 cells by Prdx2-siRNA-LV transduction. The expression of Prdx2 at both mRNA and protein levels in HCT116 cells transfected with the Prdx2 siRNA was significantly lower than that of negative
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control siRNA, as confirmed by quantitative real-time PCR and Western blotting analysis, respectively (p \ 0.05, Fig. 2a–c). VEGF-VEGFR2 axis has a critical role in VM formation, VEGF plays a crucial role in the formation of VM in an autocrine or paracrine manner, so recombinant human VEGF induction was used to mimic the microenvironment in vitro. We found that the numbers of the tubular structures were significantly decreased in Prdx2 siRNA explants than those of negative control siRNA explants after inducing by VEGF (p \ 0.01, Fig. 3a–c). The result indicates that Prdx2 knockdown impedes the VEGF-induced VM formation. The expressions of VEGFR2 and phospho-VEGFR2 were assayed by Western blotting in HCT116 cells transduced with Prdx2 siRNA and negative control siRNA after induced by VEGF at different time periods of culture. Although VEGFR2 protein expressions had no change, we found that VEGFR2 phosphorylation levels induced by VEGF were markedly reduced in cells transduced with Prdx2 siRNA compared with those of negative control siRNA by Western blotting analysis over time (p \ 0.05, p \ 0.01, Fig. 3d, e). These data indicate that Prdx2 knockdown inhibits VEGF-induced VM formation by reducing the phosphorylation level of VEGFR2. Knockdown of Prdx2 inhibits VEGF-induced invasion in HCT116 cell in vitro The effects of Prdx2 knockdown on the invasive capabilities of HCT116 cells with VEGF induction were examined by Matrigel invasion chamber assay. After VEGF induction, the invasive capabilities of HCT116 cells were significantly decreased in Prdx2 siRNA explants than those of negative control siRNA explants (p \ 0.05, Fig. 4). These data indicate that Prdx2 is a critical element of invasion in CRC progression and plays an important role in the progression of CRC.
Fig. 2 Prdx2 expression is suppressed by Prdx2 siRNA in HCT116cells. a Prdx2 mRNA expression in HCT116 cells transfected with Prdx2 siRNA is significantly reduced than that of negative control siRNA, as determined by qRT-PCR analysis (p \ 0.05). b,
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Knockdown of Prdx2 inhibits tumor growth in vivo Lastly, we examined the effect of Prdx2 deficiency on pathological tumor growth using a tumor xenograft model in vivo. After implant of HCT116 cell lines that transduced with Prdx2 siRNA and negative control siRNA as xenografts into nude mice, the growth of xenograft tumors with Prdx2 siRNA was much slower than that of negative control siRNA. After growing for 5 weeks, the volumes of tumor xenografts with Prdx2 siRNA were smaller than those of negative control siRNA (p \ 0.05, Fig. 5). These data indicate that Prdx2 Knockdown can retard tumor growth in CRC.
Discussion CRC is still the main tumor threatening to people’s health, it is vital to explore new molecular pathways as its incidence increases year by year [16]. Our team had previously reported that Prdx2 expression was elevated in CRC tissues and was significantly associated with tumor metastasis and the TNM stage (7). Prdx2 expression was closely related to the low degree of tumor differentiation and status of lymph node metastasis (22). Here, we detected Prdx2 expression in more CRC tissues than before and confirmed that Prdx2 overexpression existed in CRC tissues. To the best of our knowledge, it had been proved that VM-positive tumor patients also had more risks of metastasis and recurrence, and VM-positive expression was closely related to the low degree of tumor differentiation (21). Since Maniotis first confirmed the existence of VM in uveal melanoma, VM had been identified successively in many tumors, including gastric stromal tumor [17], ovarian cancer [12], prostate cancer [18], liver cancer [19], breast cancer [20], and CRC [11]. Prdx2 expression and VM-positive expression are both closely related to low degree of tumor differentiation
c Prdx2 protein expression is suppressed in cells after transfection with Prdx2 siRNA compared with that of negative control siRNA, as shown by Western blotting analysis (p \ 0.05)
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Fig. 3 Effect of Prdx2 on the vasculogenic mimicry formation of HCT 116 cell in 3D culture. a–c After induced by VEGF165 (50 ng/ ml), VM channels are significantly reduced in siPrdx2 explants than those of negative control siRNA explants (9200, p \ 0.01). d, e After
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induced by VEGF165 (10 ng/ml), VEGFR2 expression had no change, phospho-VEGFR2 is markedly reduced over time in cells of Prdx2 siRNA compared with that of negative control siRNA, as by Western blotting analysis (p \ 0.05, p \ 0.01)
Fig. 4 Effect of siPrdx2 on the invasive capabilities of HCT 116 cells. a–c After induced by VEGF165 (50 ng/ml), the invasive capabilities of HCT116 cells are significantly decreased in Prdx2 siRNA explants than those of negative control siRNA explants (p \ 0.05, 9200)
Fig. 5 Prdx2 knockdown inhibited the growth of xenograft tumors in nude mice. a Representative xenograft tumors are shown. b Tumor volumes are monitored over time (*p \ 0.05)
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and adverse clinical outcome, and we demonstrated a critical role of Prdx2 in the formation of VM. In our study, the incidence of VM in CRC tissues was 22.86 % (16/70), which was similar to that previously reported [21]. We found that the overexpression of Prdx2 was associated with the incidence of VM in CRC tissues. Our study also provided the evidence that the poorly differentiated CRC HCT116 cells can form obvious VM channels in vitro by using a well-established 3D culture model which was similar to that previously reported [21]. Unlike tubular channels formed by HUVEC cells, the tubular channels of HCT116 cells were made up of several layers of cells which were thicker and denser than those of HUVEC cells. As a selective cytokine, VEGF can secrete with the microenvironment changes and plays a crucial role in the formation of VM [22]. VEGFR2, as the major VEGF receptor requires phosphorylation for activation, demonstrates many signaling capacities in the formation of VM channels [23–25]. It was reported that VEGFR2 was expressed in tumor cells from patients with CRC and responded to VEGF stimulation with augmented VEGFR2-mediated proliferation and tumor growth, and the activation of VEGF receptors on tumor cells could mediate tumor growth and metastasis [26]. It has been shown that Prdxs were lost the peroxidase activity by inhibitory phosphorylation on threonine or tyrosine residue [27]. Our study showed that VEGFR2 expression was not changed significantly after induced by VEGF, but VEGFR2 phosphorylation levels were much lower over time in CRC cells of Prdx2 siRNA compared with those of negative control cells. We confirmed that Prdx2 is a key stimulatory molecule in VM formation by involving in maintenance of VEGFR2 activation. Invasion and metastasis are important characters of malignant tumors and main obstacles to cure, the invasive capabilities of cancer cells are closely related to VM formation and adverse clinical outcome, and targeting the vasculature remains a promising approach for treating solid tumors [28, 29]. The effect of Prdx2 knockdown on the invasive capabilities of CRC cells with VEGF induction was examined. After VEGF induction, the invasive capabilities of CRC cells were significantly decreased in Prdx2 knockdown explants than those of negative control siRNA explants. The effect of Prdx2 deficiency on pathological tumor growth using a tumor xenograft model in vivo also showed that Prdx2 was critical for pathological tumor growth. The tumor growth in Prdx2 deficiency mice was much slower than that of negative control siRNA mice. As a special mode of blood supply in addition to angiogenesis, VM is closely related to tumor cell growth activity. The growth and metastasis of cancer can be retarded by interfering with the formation of VM vessels [30]. We think that Prdx2 deficiency can retard CRC cells growth partly due to reduced VM vessels in vivo, but further research of mechanism is needed.
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In summary, one of our important findings is that as a major antioxidant enzyme, Prdx2 mediates VM formation by regulating VEGFR2 activation, which now represents as a therapeutic target for CRC. Acknowledgments This study was supported by a Grant from the National Natural Science Foundation of China (No. 81172295). Conflict of interest interest.
All the authors indicate no potential conflict of
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