Introduction
Colorectal cancer (CRC) is a major international health problem that is the third most frequent type of cancer and the second most common cause of cancer related death in the Western world (1). It has been reported that when CRC is diagnosed at early stage, nearly 90% of the patients can be cured by surgery. However, this disease is very often diagnosed at an advanced stage, resulting in poor prognosis subsequently (2–4). In addition, the mechanisms of CRC development and progression are not quite clear and have yet to be further explored. Therefore, more insight and new methods to investigate the underlying mechanisms of CRC are needed to identify effective biomarkers and this is critical for proper control of CRC.
In recent years, proteomics have burst onto the scientific scene rapidly (5). Based on 2-DE and mass spectrometry, hundreds of proteins can be identified simultaneously and precisely through high-throughput identification. Therefore, proteomics have been widely applied to search for diagnostic biomarkers in early disease detection, as well as mechanism analysis of disease, especially in the field of cancer research (6–8). In the present study, differentially expressed proteins between individually matched CRC and normal tissues were profiled from 8 CRC patients. Of 36 proteins identified, carbonic anhydrase II (CA II) was chosen for verification and function and mechanism analysis. It was expected that the results from the study may contribute to a better understanding of the molecular mechanism of CRC and provide insight into colorectal cancer treatment.
Materials and methods
Patients and tissue preparation
For proteomic analysis, 8 cases of CRC and pared adjacent normal tissues were obtained from West China Hospital, Sichuan University. The clinical characteristics of the patients are summarized in Table I. Fresh tissues samples were obtained immediately after the surgery, snap-frozen immediately in liquid nitrogen and then stored at −80°C before analysis. For the validation studies, 25 cases of paraffin-embedded primary CRC tissues and pared adjacent normal tissues were collected consecutively from patients at West China Hospital in 2009. Written informed consent was obtained from all patients and the study procedures were approved by the Scientific and Ethics Committee of Sichuan University (Chengdu, China).
Proteomic analysis and protein identification
2-DE was carried out as previously described (9) with minor modifications. Briefly, tissue sample was ground into powder in liquid nitrogen and sonicated in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, 0.2% ampholyte pH 3.0–10.0; Bio-Rad, Hercules, CA, USA) containing protease inhibitor cocktail. IPG strips loaded with 1 mg protein (17 cm, pH 3.0–10.0, non-linear; Bio-Rad) were passively rehydrated for 12–16 h. Having been separated according to their pI for the first dimension, the strips were transferred to the second dimension 12% SDS-PAGE for the separation according to the molecular weight. Spots that showed consistent and significant differences (>2-fold) were selected for mass spectrometry (MS) analysis.
In-gel digestion of protein was conducted using MS-grade Trypsin Gold (Promega, Madison, WI, USA) by following the manufacturer’s instructions. ESI-Q-TOF MS/MS analysis and protein identification were performed as described in our previous proteomic studies (9). Briefly, peptide mass maps were acquired using a Q-TOF mass spectrometer (Micromass, Manchester, UK) fitted with an ESI source. For MASCOT analysis, peptide and fragment mass tolerance were set at 0.1 and 0.05 Da, respectively.
Semiquantitative RT-PCR
Total RNA extraction was performed using TRIzol reagent (Invitrogen). cDNA was then synthesized using the ExScript™ reagent kit (Takara, Shiga, Japan) following the manufacturer’s instructions. The primer sequences and the expected sizes for PCR products were as follows: CA II, 5′-GTCCCATAGTCTGTATCCAA-3′ (sense) and 5′-GAGTGCTCATCACCCTACAT-3′ (antisense) (301 bp); GAPDH, 5′-TGGAAGGACTCATGACCACA-3′ (sense) and 5′-GCTTCCCACCTTCTTGATG-3′ (antisense) (280 bp). The amplification parameters consisted of 25 (CA II) or 20 cycles (GAPDH) at 94°C for 30 sec, 60°C (CA II) or 57°C (GAPDH) for 30 sec and 72°C for 30 sec. The PCR products were analyzed by electrophoresis in 1.2% agarose gels and visualized by Gold View (Takara) staining.
Western blotting
CRC tissues and cells were lysed with cold RIPA lysis buffer containing protease inhibitors. Thirty micrograms of protein extraction were applied to 12% SDS-PAGE gels and then transferred to polyvinylidene fluoride membrane. The membrane was probed with primary antibodies against CA II (1:1,000, GeneTex), E-cadherin (1:1,000, Cell Signaling Technology, MA, USA), vimentin (1:1,000, Cell Signaling Technology), PKM2 (1:1,000, Cell Signaling Technology) and GAPDH (1:1,000, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), respectively. Blots were developed with HRP-conjugated secondary antibodies (1:5,000, Santa Cruz) and chemiluminescent substrate (Millipore, MA, USA) on Kodak X-ray film.
Immunohistochemistry and immunocytochemistry
Tissue slides or SW480 cells fixed in polystyrene culture were stained with the rabbit anti-human CA II antibody (diluted 1:200, GeneTex) using the DAB substrate solution according to the manufacturer’s instructions.
Cell culture and establishment of a stable cell line
Four human colorectal cancer cell lines, SW480, SW620, HCT116 and LoVo cell were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Cells were grown in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 2 mM L-glutamine, 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained in a humidified environment containing 5% CO2 at 37°C.
For establishment of the stable cell line SW480, cells were transfected with DNA constructs (OriGene) encoding EGFP-CA II (SW480-CA II-over) or EGFP (SW480-control). Forty-eight hours after transfection, cells are harvested, diluted (1:10) and plated in fresh medium containing G418 (800 μg/ml, Invitrogen, Carlsbad, CA, USA). Colonies with green fluorescent signal were then picked and expanded.
Drug treatments and MTT assay
Tumor cells were seeded in 96-well plates at 5×103 cells per well. After 16 h, cells were incubated with various concentrations of drugs. SW480 cells were treated with different concentration of oxaliplatin (10, 20, 30, 40 and 50 μM respectively, Sigma, St. Louis, MO, USA); and, after pretreatment with 100 μM acetazolamide (Sigma) for 8 h, HCT116 cells were treated with oxaliplatin as in SW480 cells. Forty-eight hours later, the effects of drugs on cells were assessed using MTT methods. Briefly, cells were incubated with 20 μl of MTT reagent (20 mg/ml) for 4 h, followed by addition of 100 μl of solubilization solution into each well. The plates were left in the dark room overnight and optical density (OD) was measured at 590 nm wavelength. Results are expressed as percentage of viable cells compared with untreated cells (with 100% viability). The results are based on three independent experiments. Drug concentrations that inhibit 50% of cell viability (IC50) for oxaliplatin were determined using the method described previously (10).
Colony formation assay
SW480 cells overexpressing CA II and control cells were seeded at 300 cells/well in a 6-well plate with triplicate wells for each group. After 14 days of culture, cells were fixed in methanol for 30 min and stained with Giemsa (Beyotime). The number of clones consisting of >50 cells was counted. The colony forming efficiency was calculated according to the formula: (the clone number/the plated cell number) × 100.
Flow cytometry
The cells were harvested, washed twice with PBS and fixed in 70% ethanol overnight. After incubation with RNAse A and propidium iodide (Beyotime) for 30 min at 4°C in the dark, cell cycle data were collected on a flow cytometer with a 488-nm laser and analyzed with the manufacturer’s software.
Statistical analyses
All quantitative data were expressed as mean ± SD. Comparisons between two groups were performed by Student’s t-test. Statistical calculations were performed with SPSS 11.0.0 statistical software. Data were considered as statistically significant at P<0.05.
Results
Identification of differentially expressed proteins between CRC and the corresponding normal tissues
The proteome of individual-matched CRC and normal colorectal tissues from 8 patients (mean age 66.25±10.39 years; range 53–84 years) were compared by 2-dimensional gel electrophoresis (2-DE) using a broad pH gradient (pH 3.0–10.0 non-linear). Coomassie staining of 2-D gels visualized 852±46 and 871±34 protein spots within normal colorectal tissues and CRC, respectively. Representative 2-DE maps are showed in Fig. 1A and spot no. 13 (boxed in Fig. 1A) as a selected example, was significantly downregulated in CRC as shown in enlarged form in Fig. 1B. As a result, 52 spots showed >2.0-fold change (P<0.05). Differentially expressed protein spots were subsequently subjected to MS/MS analysis. Of 52 spots, 44 spots corresponding to 36 unique proteins were identified probably due to post-translational modification such as protein phosphorylation (Table II). Notably, carbonic anhydrase II (CA II) corresponding to spot no. 13 (Fig. 1B) was found to be one of the most significantly differential expression between cancer and normal tissues. It was downregulated >5-fold in CRC compared with the normal tissues. The mass spectra of CA II is shown in Fig. 2. MS/MS analysis of CA II revealed 8 matched-peptides, 50% sequence coverage and a MOWSE score of 226. Due to the confident identification, CA II was chosen as the subsequent focus of this study.
Validation of CA II by semiquantitative RT-PCR and western blot analysis
To confirm the differential expression of CA II between CRC and corresponding normal tissues, validation experiments were performed by RT-PCR and western blot analysis at mRNA and protein level, respectively. The result of RT-PCR analysis showed significantly different mRNA level of CA II between CRC and normal tissues (cancer tissues, 0.31±0.07; normal tissues, 0.98±0.25; Student’s t-test, P<0.01) (Fig. 3A). Western blot analysis was performed using anti-CA II antibody and remarkable CA II downregulation was observed in CRC tissues (cancer tissues, 0.22±0.05; normal tissues, 0.85±0.28; Student’s t-test, P<0.01) (Fig. 3B). Taken together, our data demonstrated that CA II expression notably decreased in CRC compared with normal tissues, which was consistent with the results of 2-DE.
Further verification of CA II expression by immunohistochemistry
To further confirm the reduction of CA II expression in CRC, 25 paraffin-embedded individual-matched CRC and normal colorectal tissues were stained using anti-human CA II antibody. Strong positive staining for CA II mainly located in the cytoplasm and nucleus of epithelium and gland cells in normal colorectal tissues. In contrast, there were weakly or negative staining signal in CRC tissues (Fig. 3C). As shown in Table III significant differences in staining intensity and positive cells were observed between CRC and normal colorectal specimens (rank-sum test, P<0.05). The semiquantitative scoring of immunoreactivity for normal tissues and CRC was 6.45±2.84, 1.57±0.86, respectively (Student’s t-test, P<0.01), suggesting the expression of CA II had a decreased tendency in both frequency and intensity from normal tissues to CRC.
CA II overexpression exerts inhibitory effect on CRC cell growth both in vitro and in vivo
In order to investigate the function of CA II in colorectal carcinoma, CRC cancer cell line SW480 was used to establish a stable cell line overexpressing CA II (SW480-CA II-over), since CA II was not detected in SW480 cells (Fig. 4A). Western blot and immunocytochemistry analysis showed that in contrast with no expression in control stable cell line (SW480-control), remarkable expression of CA II was observed in SW480-CA II-over stable cell line (Fig. 4A and B), suggesting successful establishment of stable SW480-CA II-over cell line.
To examine the effect of on CRC cells, MTT assay was carried out. As shown in Fig. 5A, CA II overexpression notably suppressed SW480 cell viability in a time-dependent manner. Colony formation assay showed that overexpression of CA II in SW480 cell significantly suppressed colony formation efficiency compared with control cells (Fig. 5B). Further flow cytometry analysis demonstrated that SW480 cells stably and highly expressing CA II were stalled at G0/G1 and G2 phase with subsequent decrease in S phase compared with control stable cell line (Fig. 5C).
Moreover, the inhibitory effects on CRC cancer cell growth were examined in an animal model. Tumor growth curve drawn based on the data from in vivo tumor model showed that SW480 overexpressing CA II had a slowed growth rate (Fig. 5D). Representative images of tumor dissection showed that high expression of CA II in tumor cell resulted in suppressed tumor volume (Fig. 5E). As shown in Fig. 5F, there was a remarkable difference in average tumor weight between CA II-overexpression and control group (CA II-over, 301.3±120.7 mg; control, 730±240.5 mg; Student’s t-test, P<0.01). Our results in vitro and in vivo suggested that CA II could serve as a tumor suppressor gene and suppress colorectal carcinoma growth and development.
Cytotoxicity assay suggests CA II increases the sensitivity of CRC cells to oxaliplatin
Through catalyzing the reversible reactions of CO2 and water: CO2 + H2O⇆H+ + HCO3−, CA II exerts an important role in acid-base balance in living organism. Thus, it was hypothesized that some signal pathways underling tumor development might be regulated by CA II. As a rate-limiting enzyme of aerobic glycolysis in tumor, embryonic M2 isoform of pyruvate kinase (PKM2) was first determined by western blotting. Similar protein level of PKM2 was observed in control and SW480-CA II-over cells (Fig. 6A); there was no significant difference of PKM2 expression in CRC and corresponding normal tissues (Fig. 6B). E-cadherin and vimentin, two markers corresponding to epithelial and mesenchymal cells, respectively, in epithelial-mesenchymal transition (EMT) were then examined. As shown in Fig. 6A and B, both in vitro cells and CRC tissues, no differential expression of CA II was observed.
Since extracellular acidification of cancer cells in tumor tissues leading to decreased anticancer drug uptake is one of drug-resistance mechanism, it was hypothesized that through pH regulation, CA II may affect sensitivity of CRC cancer cells to anticancer drug. Chemosensitivity tests by MTT showed that CA II overexpression increased the sensitivity of SW480 cells to oxaliplatin (IC50), from 20.5±4.3 μM in control group to 11±5.5 μM (Student’s t-test, P<0.05). In contrast, for HCT116 cells with high expression of CA II, with or without pretreatment with acetazolamide, a non-specific antagonist against CA II, the corresponding IC50 for oxaliplatin was 30.3±3.2 and 23±4.1 μM, respectively (P<0.05), suggesting CA II could induce the chemotherapeutic sensitivity of colon cancer cells.
Discussion
In the Western world colorectal cancer (CRC) is the third most frequent type of cancer and the second most common cause of cancer related death (1). Clearly, early diagnosis and prognosis are urgent for efficient control of CRC and this largely dependents on more advances in the knowledge of mechanisms associated with CRC. In the present study, we compared the proteome between CRC and corresponding normal tissues with 2-DE and MS/MS-based approach. Thirty-six differentially expressed proteins were identified between two groups and most of these proteins were involved in fundamental biological processes. Among these 36 proteins, CA II was further studied according to the following selection criteria: i) it is one of the most significantly and differentially expressed proteins between CRC and matched normal tissues; ii) good reliability of the MS identification of protein; iii) evolutionarily conserved sequence and physiological function is crucial; iv) no or few studies reported the function and mechanism in tumor.
Carbonic anhydrase II (CA II), one of CA family isozymes, catalyzes the reversible hydration of carbon dioxide: CO2 + H2O⇆H+ + HCO3−, which is involved in many critical physiological or biochemical processes based on ion transport and pH balance such as respiration, digestion, bone resorption and renal acidification (11). In addition to physiological function, it has recently been found that CA II is abnormally expressed in many types of human cancer. It was noteworthy that there was no consistent expression profile of CA II in different types of cancer tissues (12–15). Moreover, there is a contradictory correlation between CA II and the prognosis, progression of cancer patient among different types of cancer (12–15). It was shown that CA II was overexpressed in most gastrointestinal stromal tumors and strong staining of CA II indicated significantly better survival rates, suggesting CA II may serve as a diagnostic and prognostic biomarker for gastrointestinal stromal tumors (12). In contrast, immunostaining of the tumors and normal tissues from melanoma, esophageal, renal and lung cancers revealed that CA II was expressed in the tumor vessel while not in normal vessel endothelium (13). Furthermore, compared with negative staining, positive staining of CA II in vessel endothelial cells from meningiomas and glial tumors predicted worse survival rates (14,15). In our study, comparative proteomic analysis showed expression of CA II notably decreased in CRC compared to normal colorectal tissues. RT-PCR, western blot analyses and immunohistochemistry were further performed to validate downregulation of CA II in CRC tissues and these results were also consistent with previous studies that paralleled with increasing severity of colorectal tissue lesions and progression of CRC, the staining intensity of CA II among normal tissues, benign lesions and malignant lesions revealed clearly decreased tendency (16–18). However, function and mechanism of CA II in the CRC development and progression have not been investigated.
Considering that CA II expressed is low in CRC tissues, gain of function strategy was utilized to study function of CA II in CRC. Stable cell line overexpressing CA II, SW480-CA II-over was then established given that CA II could not be examined in colorectal cancer cell line SW480. Serial in vitro as well as in vivo experiment results demonstrated that overexpressing CA II significantly suppressed colorectal cancer cell SW480 proliferation both in vitro and in vivo, which could be partially explained by remarkable cell cycle arrest at G0/G1 and G2 phase. To our knowledge, there is no report on how CA II functions in cancer development and progression, in spite of its abnormal expression in many types of cancer. We report that at least in colorectal cancer, CA II may play a role as tumor suppressor gene in cancer development and progression.
A distinguishing phenotype of acidic extracellular pH (pHe) and alkaline intracellar pH (pHi) in solid tumors appears to give selective advantage for tumor growth and development (19). Since carbonic anhydrase isoenzymes were involved in generating acidic tumor microenvironment (20,21), we hypothesized that CA II may also influence the processes associated with tumor microenvironment. Embryonic M2 isoform of pyruvate kinase (PKM2) was first determined by western blotting since it is a rate-limiting enzyme of aerobic glycolysis in tumors and this is specific to metabolism of solid tumors originally described by Otto Warburg (22,23). Overexpressing CA II failed to alter PKM2 protein level and there was no significant change of CA II between matched CRC and normal tissues. Epithelial-tomesenchymal transition (EMT) is a transdifferentiation shift in which epithelial cells lose adhesiveness and polarity and acquire spindle morphology and migratory capacity characteristic of fibroblasts (24,25). It has been shown that EMT plays crucial roles in acquisition of tumoral invasiveness, the initial step of the metastatic cascade in cancer (24,25). Differently expressed E-cadherin and vimentin, two markers corresponding to epithelial and mesenchymal cells, respectively, were not observed in vitro or in tissues. Changed tumor microenvironment can influence the uptake of anticancer drugs and modulate the response of tumor cells to anticancer drugs (19). In the present study, overexpression of CA II decreased the oxaliplatin IC50 compared with that in control SW480 cells. In contrast, in HCT16 cells with high CA II expression, oxaliplatin IC50 increased after pretreatment with CA II antagonist, which suggested that CA II could increase the sensitivity of colorectal cancer cells to chemotherapy drugs. Inconsistent with our results, Mallory et al (26) found that in highly tumorigenic MDA-MB-231 breast cancer cells, knockdown of CA II expression using RNAi strategy resulted in less IC50 than in control cells for doxorubicin, an antineoplastic drug, implicating that CA II may negatively regulate sensitivity of breast cancer cells to chemotherapy drugs. In order to explain this contradiction and get more precise results, it is obviously necessary to increase the number of cell types and chemotherapy drugs for IC50 determination.
In the present study, we only utilized gain of function strategy to study the functions of CA II. Through establishing the colorectal cancer cell line stably overexpressing CA II, we concluded that CA II might serve as a tumor suppressor gene in at least CRC development and progression. In future research, it is necessary to further strengthen our conclusion by using loss of function strategies such as knockout or knockdown of CA II gene expression. Another limitation is the mechanism by which CA II suppressed the development and progression of CRC has yet to be thoroughly revealed, although it was shown that CA II could increase the sensitivity of CRC cells to anticancer drugs. Further study to explore the mechanism of CA II tumor inhibitory effects will be conducted in our research group.
In this study, CA II was identified by proteomic analysis as a potential biomarker for diagnosis of CRC followed by further verification by molecular biology methods. Moreover, it was shown that CA II might play a role as tumor suppressor gene in cancer development and progression. In conclusion, our data may contribute to a better understanding of the molecular mechanism of CRC and provide insight into colorectal cancer treatment.