The authors have declared that no competing interests exist.
Conceived and designed the experiments: LJ. Performed the experiments: XX JR YYT. Analyzed the data: LJ. Contributed reagents/materials/analysis tools: HQW QY MJW. Wrote the paper: WT.
In order to understand the molecular mechanisms of Bifidobacterium infantis thymidine kinase/nucleoside analogue ganciclovir (BI-TK/GCV) treatment system which was proven to exhibit sustainable anti-tumor growth activity and induce apoptosis in bladder cancer, a proteomic approach of isobaric tags for relative and absolute quantification (iTRAQ), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used. 192 down-regulated and 210 up-regulated proteins were identified after treatment with BI-TK/GCV system in Sprague-Dawley (SD) rats. Western blot analysis and immunohistochemistry analysis confirmed that Peroxiredoxin-I (Prx-I) was significantly down-regulated in bladder cancer after treatment. Prx-I silencing by transfection of Prx-I shRNA significantly suppressed growth, promoted apoptosis and regulated the cell cycle in T24 cells and reduced the phospho-NF-κB p50 and p65 protein expression which revealed the links between Prx-I and NF-κB pathway implied by Ingenuity pathway analysis (IPA). These findings yield new insights into the therapy of bladder cancer, revealing Prx-I as a new therapeutic target and indicating BI-TK/GCV system as a prospective therapy by down-regulation of Prx-I through NF-κB signaling pathway.
Bladder cancer is the most common urological cancer in Asia and its clinical management is extremely expensive
Herpes simplex virus thymidine kinase (HSV-TK)–mediated suicide gene therapy as a widely accepted strategy for bladder cancer can convert the nontoxic nucleoside analog ganciclovir (GCV) into a toxic triphosphorylated form, which subsequently causes the death of rapidly dividing cells
In an effort to understand the underlying molecular mechanisms and identify the potential target protein molecule of this safe and effective treatment system, we resorted to mass spectrometry (MS)-based isobaric tags for relative and absolute quantification (iTRAQ) to obtain comprehensive differential protein profiles. Furthermore, we investigated the molecular pathway of Peroxiredoxin-I (Prx-I), one of the identified down-regulated proteins in bladder cancer identified by iTRAQ after treatment with BI-TK/GCV.
The BI-TK/GCV treatment system was constructed successfully by our research group (Chongqing, China)
Seventy female Sprague–Dawley rats (6–8 weeks old, weighing 180–200 g) were purchased from Chongqing National Biological Industry Base of Experimental Animal Center (China) and housed under specific pathogen-free condition at 23–27°C and humidity 55–65% with 12-h light–dark cycles. All animal procedures were approved by the Animal Use and Care Committee of Chongqing Medical University. A rat bladder tumor model was built by perfusion of N-methyl-nitrosourea (MNU)(Sigma, USA). MNU was diluted into 20 g/l by a citric acid buffer solution. Each bladder was perfused with 0.1 ml once every 2 weeks, for a total of four perfusions.
Sixty tumor-bearing Sprague-Dawley rats were randomly divided into four groups (each
The tissues were lysed in a lysis buffer (7 m urea, 1 mg/ml DNaseI, 1 mmNa3VO4, and 1 mm phenylmethane sulfonylfl uoride, PMSF) and centrifugated at 6000 g and 4°C for 30 min. The supernatant was collected and the total protein content was measured using 2D Quantification Kit (Amersham Biosciences, Uppsala, Sweden). Each sample was digested with 20 µl of 0.1 µg/µl trypsin solution (Promega, Madison, USA) at 37°C overnight and then labeled with the iTRAQ tags as follows: (i) normal saline group, 114 tags; (ii) BI-TK group, 115 tags; (iii) BI/PGEX-1 group, 116 tags; (iv) BI group, 117 tags. The labeled samples were pooled prior to further analysis.
To reduce the sample complexity for liquid chromatography (LC)-MS/MS analysis, the pooled samples were diluted 10-fold with an SCX buffer A (10 mm KH2PO4 in 25% acetonitrile at pH 3.0) and utilized to a 2.1×200 mm polysulfoethyl A SCX column (PolyLC; Columbia, USA). The column was eluted with a gradient of 0–25% SCX buffer B (10 mM KH2PO4 at pH 3.0 in 25% acetonitrile containing 350 mM KCl) over 30 min, followed by a gradient of 25–100% SCX buffer B over 40 min. These SCX fractions were lyophilized in a vacuum concentrator and subjected to C-18 clean-up using extraction column (100 mg capacity, Supelco; Sigma-Aldrich, St. Louis, USA).
MS was performed using a nano-LC coupled online to a QStar Elite mass spectrometer (Applied Biosystems). The LC eluent was directed to an ESI source for Q-TOF-MS analysis. The mass spectrometer was set to perform information dependent acquisition (IDA) in the positive ion mode, with a selected mass range of 300–2,000 m/z. Peptides with +2 to +4 charge states were selected for tandem mass spectrometry, and the time of summation of MS/MS events was set to 3 s. Relative quantification of proteins, in case of iTRAQ, was performed on the MS/MS scans and was the ratio of the areas under the peaks at 113, 114, 115, and 116 Da, which were the masses of the tags that correspond to the iTRAQ reagents.
The bioinformatic processes and molecular function of the identified proteins in the BI-TK group after treatment were classified by the PANTHER classification system (
Western blotting and IHC were used to confirm the expression of Prx-I. T The protein samples (about 20 mg) were separated using SDS–PAGE. After SDS–PAGE electrophoresis, proteins were transferred to PVDF membranes. Subsequently, the lysates were incubated with a primary anti-Prx-I rabbit monoclonal antibody (1∶1500) (Abcam, USA). The immunoreactive signals were detected by enhanced chemiluminescence kit (Amersham Biosciences, Sweden). The procedures were conducted according to the manufacturer's instructions. Bladder cancer tissues from four groups were incubated overnight with primary antibodies. The tissues were incubated with secondary antibodies for 2 h. The cell nuclei were counterstained with hematoxylin. The proportion of positively stained tumor cells was determined by Image-Pro Plus (IPP) 6.0 and was graded as follows: 0, negative; 1, <10%; 2, 10–50%; 3, >50%. The immunostaining intensity was scored as follows: 0, absent; 1, light yellow; 2, yellowish brown; 3, brown. The protein in bladder cancer tissues was evaluated using the staining index (SI):
T24 cells were subjected to Prx1 knockdown. Short hairpin RNA (shRNA) was exprssed with GV102 system (GeneChem, China). The three pairs of sense and antisense sequences of oligonucleotides targeting human Prx1(GeneBank_ID: NM_002574) were as follows: PRDX1-RNAi (sh-1) sense strand
After transfection with Prx-I shRNA for 24 h, the T24 cells were seeded into 96-well culture plates at a density of 4×103 cells in a final volume of 100 µl/well, and the untransfected cells were used as a control. The cell proliferation rate was calculated at different time points (24, 48 and 72 h) using Cell Counting Kit-8 (CCK-8) (Sigma, USA). Experiments were performed according the manufacturer's protocol. The absorbance at 450/630 nm was measured with a Thermo spectrophotometer (Waltham, USA). The average absorbance from six wells per group was calculated.
After transfection for 48 h, the cells were trypsinized and centrifuged at 1500 rpm for 5 min. The cells were harvested and washed with PBS twice. After stained with 50 µg/ml Annexin V-fluorescein isothiocyanate (FITC) (BD Biosciences, USA) and 20 µl of 500 µg/ml propidium iodide (PI) (Sigma, USA) for apoptosis detection, the cells were incubated in dark at room temperature for 15 min and subjected to flow cytometry analysis (FACS). Then the cells were collected, washed with PBS, fixed with 75% ethanol at −20°C overnight. The fixed cells were washed with cold PBS twice, added 500 µL DNA staining solution (including 200 µg/mL RNase A and 20 µg/mL propidium iodide staining solution) and incubated for 30 minutes. Finally, the cells were subjected to cell cycle analysis by FACS. The data were analyzed and evaluated on the program ModFit (Topsham, USA).
The link between Prx-I and the NF-kappa-B (NF-κB) complex signaling had been implied in the protein pathway of IPA. Therefore, in order to explore whether the effect of Prx-I on apoptotic signaling proteins was attributable to NF-κB inhibition, we evaluated nuclear levels of phospho-NF-κB p50 and p65 (Abcam, USA) by Western blot.
The data were expressed as mean ± standard deviation (SD) and compared using analysis of variance. The level of significant difference was defined as p<0.05. All analyses were performed on SPSS 18.0 (SPSS, Chicago, USA) for Windows.
A total of 2343 unique proteins were identified with 95% confidence by the ProteinPilot search algorithm against the IPI rat protein database v3.49. A strict cutoff value of a 1.3-fold change resulted in a final set of 402 differentially expressed proteins, including 192 down-regulated proteins and 210 up-regulated proteins in the BI-TK group after treatment. Strikingly, a novel molecule Prx- I drew our particular attention, with a 0.52-fold decrease in the BI-TK group versus the normal saline group. A schematic diagram of iTRAQ is shown in
A: Schematic diagram showing the workflow of iTRAQ. B: MS/MS spectrum showing the peptides of Prx-I (peptide sequence: VVGGDHVEVHAR). The 4 peak contours describe that the sample volumes are the same which guarantees the results are authentic and reliable.
To probe into their biological roles in the curative effect of BI-TK/GCV on bladder cancer, the differentially expressed proteins were categorized into various processes and function classes based on PANTHER classification system. In biological process analysis, the largest proportion of differentially expressed proteins was in metabolic process, followed by cellular process and cell communication process (
PANTHER classification of proteins based on (A) Biological process and (B) molecular function. (C) Interplaying network of proteins with abundance change generated by Ingenuity pathway analysis (IPA). The network implied the connection of Prx-I and NF-κB complex.
The differential expression levels of Prx- I identified by iTRAQ approach were validated by Western blot (
A: Expressions of Prx-I in four groups by Western blot analysis. Beta-actin was used as a loading control. B: Representative images showing the immunoexpression of Prx-I in tumor tissues of four groups. Compared with the normal saline group, expression of Prx-I is down-regulated in the other three groups (especially in the BI-TK group), which is similar to the results obtained by iTRAQ. (Asterisk (*) indicates P<0.05 in BI-TK group versus normal saline group)
First, the Prx-I mRNA levels from four shRNA vectors transfected for 48 h in the T24 cell lines were measured by qPCR using Lipofectamine 2000. The Prx-I expression decreased by ∼40%, 26%, 18% and 1% in the sh-1, sh-2, sh-3 and con sh groups, respectively compared to the parental group (
A: The expression of Prx-I mRNA was examined by qPCR. GAPDH served as an internal control. B: The Prx-I protein levels were analyzed by Western blot after transfection. Sh-1 treatment led to a significant reduction in Prx-I protein expression in T-24 cells. (Asterisk (*) indicates P<0.05 in sh-1 group versus parental group)
The effects of Prx-I shRNA transfection on T24 cell growth were investigated through CCK8 assays. A slight inhibition in growth was observed at 24 h after transfection. Furthermore, obvious inhibitory effects on cell proliferation were observed in Prx-I knockdown-cells at 48 and 72 h, compared with the parental and con sh groups (
The growth rates in Prx-I knockdown group was significantly reduced, compared with the parental and con sh groups, measured by CCK8 assay.
The effects of Prx-I knockdown on apoptosis and cell cycle of T24 cell were investigated. After 48 h of transfection, the apoptosis rate in the sh-1 group (21.99±1.10%) was significantly higher compared with the con shRNA group (4.51±0.73%) and parental group (4.96±0.46%) (P<0.05) (
A: Prx-I knockdown induced apoptosis in T24 cells. B: Representative pictures of FACS analysis showing Prx-I knockdown induced G0/G1 cell cycle arrest in T24 cells with a corresponding decrease in S-phase cells (P<0.05).
As described above, Prx-I is directly linked to NF-κB complex, which has been implied in the protein pathway (
A significant decrease in the protein expression of both phospho-NF-κB p50 and p65 in sh-1 group. (Asterisk (*)indicates P<0.05 in sh-1 group versus parental group)
In our previous study, BI-TK/GCV was constructed and proved effective in inhibiting the progressive growth of bladder tumor which was related to apoptosis in vivo
In this study, a proteomic approach iTRAQ was used to identify differentially expressed proteins, aiming to reveal the molecular mechanisms and provide theoretical support for the effectiveness of BI-TK/GCV system. iTRAQ identified 402 differentially expressed proteins in bladder cancer tissues after treatment, including 192 downregulated proteins and 210 upregulated proteins. The targeting proteins with differential abundance (
Prx-I stood out in our proteomic analysis because it had rarely been linked directly with bladder cancer although it has been shown to down-express in bladder cancer tissues after treatment with BI-TK/GCV. The mammalian peroxiredoxin (Prx) family which consists of six proteins is H2O2-scavenging enzymes present in procaryotic and eucaryotic cells
Fortunately, the links between Prk-I and the NF-κB complex signaling have been implied in the protein pathway of IPA (
Taken together, we identified that Prx-I along with the NF-κB pathway contributed to bladder cancer for the first time. The BI-TK/GCV treatment system exhibited a sustainable anti-tumor growth activity and induced apoptosis in bladder cancer tissues by inhibition of Prx-I through the NF-κB pathway. Our research provides a new insight into bladder cancer treatment and indicates that BI-TK/GCV treatment system by targeting at Prx-I can be a novel therapeutic strategy in the future.
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