Functional characterization of the nitrogen permease regulator-like-2 candidate tumor suppressor gene in colorectal cancer cell lines.

The nitrogen permease regulator‑like‑2 (NPRL2) gene is a candidate tumor suppressor gene, which has been identified in the 3p21.3 human chromosome region. Decreased expression levels of NPRL2 have been observed in colorectal cancer (CRC) tissues, however, the function of NPRL2 in CRC progression remains to be fully elucidated. The present study investigated the biological characteristics of the HCT116 and HT29 CRC cell lines overexpressing exogenous NPRL2. NPRL2 recombinant lentiviral vectors were also constructed and transfected in the present study. Cell growth was determined using a Cell Counting Kit‑8 assay and a colony formation assay. The cell cycle and rate of apoptosis were assessed using flow cytometric analysis. Transwell assays were used to evaluate cell invasion. The protein expression of phosphorylated (p)‑AKT and caspase 3, B‑cell lymphoma 2 (Bcl2) and Bcl‑2‑associated X protein apoptosis‑associated genes, were detected using western blotting. The results revealed that NPRL2 overexpression inhibited cell growth, induced cell cycle G1 phase arrest, promoted apoptosis and inhibited invasion in the two human CRC cell lines. Furthermore, the protein expression levels of p‑AKT and Bcl2 were significantly reduced in the NPRL2‑transfected HCT116 and HT29 cells, compared with the mock‑transfected group and control group, while the protein expression of caspase‑3 was increased. Therefore, NPRL2 acted as a functional tumor suppressor in the CRC cell lines.

Introduction

Colorectal cancer (CRC) is one of the most common types of cancer worldwide, with ~1,000,000 cases diagnosed each year (1). In the last two decades, the incidence of CRC has increased in China, however, the prognosis remains poor indicating the importance of clarifying the pathogenesis of CRC. Previous studies have demonstrated that the formation and development of CRC is polygenic and involves multi-stage changes, in which oncogene mutation and tumor suppressor gene (TSG) inactivation are important (2,3).

The nitrogen permease regulator-like-2 (NPRL2) gene, is one of the candidate TSGs identified in the 3p21.3 human chromosome region, in which genomic abnormalities, including loss of heterozygosity and homozygous deletion are frequently found in the early stages of the development of various types of cancer in humans (4). The downregulation or silencing of the NPRL2 gene via aberrant splicing transcripts, multiple exon deletions or intragenic homozygous deletions, has been observed in renal cell carcinoma, lung cancer and other types of cancer and cancer-derived cell lines in humans, suggesting that NPRL2 may be a tumor suppressor, the inactivation of which may promote tumori-genesis (4–8). Decreased mRNA and protein expression levels of NPRL2 have also been found in CRC tissues, compared with matched normal tissues and adenomas (9,10). However, how the functional characterization of NPRL2 contributes to the progression of CRC remains to be elucidated. Thus, the presents study investigated the biological characteristics of two CRC cell lines exhibiting overexpression of exogenous NPRL2.

Materials and methods

Cell lines

HCT116 and HT29 colon cancer cell lines, obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China), were cultured in RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS) at 5% CO2, 37°C, 95% humidity.

Construction of NPRL2 recombinant lentiviral vectors and transfection

The lentivirus overexpressing the NPRL2 gene was constructed and purchased from Shanghai Genechem Co., Ltd. (Shanghai, China). The lentiviral vector system consisted of GV208, a pHelper 1.0 vector and a pHelper 2.0 vector prior to packaging. The full length of the human NPRL2 gene (cat. no. AF040707), which was marked by enhanced green fluorescence protein (eGFP), was encoded into the GV208 vector. The three vectors were co-transfected into 293T cells obtained from the Shanghai Institute of Cell Biology (Shanghai, China) in serum-free medium using Lipofectamine 2000 (Invitrogen Life Technologies). The medium was replaced with complete medium following 8 h of incubation. High-titer recombinant lentiviral vectors carrying NPRL2 were harvested after 48 h of transfection. HCT116 or HT29 cells in the logarithmic growth phase were seeded, at a concentration of 5×105 cells/well, in 96-well plates and transfected with the NPRL2-GFP vectors or GFP lentiviral vectors (mock) in serum free medium in a 37°C incubator. Polybrene (Sigma-Aldrich, St. Louis, MO, USA) was added to improve the transfection efficiency as an enhancing reagent. After 8 h transfection, the medium was replaced with complete medium. After 3 days, the expression of GFP was examined using fluorescence microscopy (BX51; Olympus, Tokyo, Japan). Cells with stable expression of eGFP were harvested for subsequent analyses.

Western blotting

Cells were lysed on ice for 30 min with lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing 1% protease inhibitor (phenylmethanesulfonylfluoride). The protein contents of cleared lysates were analyzed quantitatively using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), following which 20 μg protein samples were separated using 5% SDS-PAGE (Sigma-Aldrich) and then transferred onto polyvinylidene difluoride (PVDF; Beyotime Institute of Biotechnology) membranes. Following blocking with a buffer containing 5% low fat milk and 0.1% Tween-20 in Tris-buffered saline. The membrane was incubated at 4°C overnight with the following monoclonal mouse anti-human primary antibodies: GAPDH (cat. no. LS-C82121), NPRL2 (cat. no. LS-C55640), phosphorylated (p)-AKT (cat. no. SC-377556), Bcl-2 -associated X protein (Bax; cat. no. LS-C88394), B-cell lymphoma 2 (Bcl2; cat. no. LS-C95461) and caspase 3 (cat. no. LS-C87555). All antibodies were purchased from Univ-Bio Inc. (Shanghai, China) and diluted 1:1,000. The membrane bound with primary antibody was subsequently incubated with a horseradish peroxidase-conjugated secondary goat anti-mouse IgG (Univ-Bio; cat. no. DC02L-200UGCN) for 2 h at room temperature. Finally, images of the blots were captured manually using a Developing and Fixing kit and an ECL substrate (Beyotime Institute of Biotechnology).

Cell growth analysis

Cell growth was determined using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Kumamoto, Japan). Briefly, ~5,000 cells were seeded onto a 96-well plate in quadruplicate for each condition. After 4 h, CCK-8 reagent was added to each well in 10 μl, and the cells were incubated for a further 5 h at 37°C. The absorbance of each sample was measured at 450 and 630 nm using a Multiskan Spectrum microplate reader (Thermo Fisher Scientific). after 0, 12, 24, 48 and 72 h of incubation.

Colony formation assay

For the colony formation assay, ~1,000 cells were seeded into six-well plates with 2 ml culture medium. Following culture in RPMI 1640 media, supplemented with 10% FBS at 37°C and 5% CO2 for 2 weeks, the cells were washed twice with phosphate-buffered saline (PBS) and stained with Giemsa (Sigma-Aldrich), following which the number of colonies containing >50 cells were counted. The cloning efficiency was calculated as follows: Cloning efficiency (%) = (number of clones / number of seed cells) × 100%. Microsoft Excel was used to construct the growth curves.

Flow cytometric analysis

The cells were harvested directly or 48 h after transfection and washed with ice-cold PBS. The propidium iodide/RNase staining kit (MultiSciences Biotech Co., Ltd., Hangzhou, China) and Annexin V-fluorescein Isothiocyanate Apoptosis Detection kit (Nanjing KeyGEN Biotech Co., Ltd., Nanjing, China) were used to assess the cell cycle and apoptosis using a FACScan instrument (BD FACSCalibur; BD Biosciences, Mountain View, CA, USA), respectively.

Transwell assays

Transwell (24-well) chambers (Costar, Cambridge, MA, USA) were used to evaluate cell invasion, according to nont-hermal plasma treatment, as described previously (11). Initially, fibronectin (2 μg/filter; Sigma-Aldrich) was dissolved in 100 μl minimum essential medium and added to the upper surface of a polyethylene filter (pore size, 8 μm). The wells were coated overnight in a laminar flow hood. Subsequently, 105 cells (in 100 μl growth medium) were added to the upper surface of the filter in the upper chamber. The Transwell chamber was incubated for 24 h in 5% CO2 at 37°C. Finally, the attached cells in the lower section were stained with hematoxylin and eosin (Beyotime Institute of Biotechnology), and counted using light microscopy.

Statistical analysis

The results for continuous variables are expressed as the mean ± standard deviation. Differences were assessed using Student's t-test. P<0.05 was considered to indicate a statistically significant difference. Analyses were performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA).

Results

Transfection efficiency of the lentivirus

To investigate the function of NPRL2, the HCT116 or HT29 cells were transfected with a lentiviral vector, which containing a NPRL2 gene encoding the overexpression of NPRL2. Cells were divided into three groups: Negative control, mock and NPRL2 overexpression. The transfected cells expressed eGFP following transfection with the lentiviral vectors. The cells were transfected by the lentiviral vectors at different multiplicities of infection (MOI), and the expression of GFP expression was examined 3 days after transfection using fluorescence microscopy. The efficiency of the transfection, calculated as the mean proportion of GFP-expressing cells of the total cell count, was >80% at a MOI of 10 (Fig. 1A). The protein expression was further confirmed using western blot analysis. The protein levels of NPRL2 were significantly higher in the NPRL2-transfected group than those in the negative control and mock-transfected groups (P<0.05; Fig. 1B).

Figure 1

Transfection efficiency of the lentivirus. (A) Monitoring of the transfection efficiency of the lentivirus into the HCT116 and HT29 cells using a light microscope and a fluorescence microscope that measured eGFP expression (magnification, x200). (B) Protein levels of the NPRL2, determined by western blotting. NPRL2, nitrogen permease regulator-like-2; eGFP, enhanced green fluorescence protein. Control, cells without transfection; mock, cells transfected with mock vector; NPRL2, cells transfected with NPRL2-overexpression vector.

Inhibition of growth by the overexpression of NPRL2

Growth curves revealed a significant deceleration of cell growth in the NPRL2-transfected group, compared with the mock-transfected and control groups in the HCT116 and the HT29 cell lines (P<0.05; Fig. 2A). In addition, the cloning efficiency in the NPRL2-transfected groups was significantly decreased (P<0.05; Fig. 2B), which indicated that the expression of NPRL2 caused the inhibition of cell growth.

Figure 2

Inhibition of growth by the overexpression of NPRL2. (A) Cell proliferation curves, determined using Cell Counting Kit-8 assays. (B) Colony formation assay, in which 1×103 cells were seeded into six-well plates and cultured in RPMI 1640 for 2 weeks. The colonies were then stained with Giemsa, counted and images were captured. Control, cells without transfection; mock, cells transfected with mock vector; NPRL2, cells transfected with NPRL2-overexpression vector. NPRL2, nitrogen permease regulator-like-2.

Effects on cell cycle and apoptosis

A flow cytometric assay was performed to analyze the effects of the overexpression of NPRL2 on the cell cycle and apoptosis in the CRC cell lines. As shown in Fig. 3, the enforced expression of NPRL2 resulted in cell cycle arrest in the G1 phase of the cell cycle and a marginal decrease in the number of cells in the S phase and G2 phases in the HCT116 and HT29 cells, compared with the mock-transfected and control groups (P<0.05). In addition, the overexpression of NPRL2 induced a marked increase in the apoptotic rate of the two cell lines (P<0.05; Fig. 4).

Figure 3

Effects of the overexpression of NPRL2 on cell cycle. Flow cytometric analysis revealed that the enforced expression of NPRL2 resulted in cell arrest in the G1 phase of cell cycle and marginal decreases in the number of cells in the S and G2 phases in the HCT116 and HT29 cells, compared with the mock-transfected and control groups. Control, cells without transfection; mock, cells transfected with mock vector; NPRL2, cells transfected with NPRL2-overexpression vector. NPRL2, nitrogen permease regulator-like-2.

Figure 4

Effects of the overexpression of NPRL2 on apoptosis. Flow cytometric analysis revealed that the NPRL2-transfected groups exhibited a marked increase in apoptosis, compared with the mock-transfected and control groups. Upper left quadrant, necrotic cells; lower left, normal cells; upper right, early apoptotic cells; lower right, late apoptotic cells. Control, cells without transfection; Mock, cells transfected with mock vector; NPRL2, cells transfected with NPRL2-overexprseeion vector. Control, cells without transfection; mock, cells transfected with mock vector; NPRL2, cells transfected with NPRL2-overexpression vector. NPRL2, nitrogen permease regulator like-2.

Effects on cell invasion

Transwell assays were performed to analyze the effects of overexpression of NPRL2 on the invasive potential of CRC cells. As shown in Fig. 5, the numbers of HCT116 and HT29 cells in the lower chamber were significantly reduced in the NPRL2-transfected group, compared with the mock-transfected and control groups (P<0.05), which indicated that the expression of NPRL2 caused inhibition of cell invasion.

Figure 5

Inhibition of invasion by overexpression of NPRL2. Transwell assays were performed to analyze the effects of the overexpression of NPRL2 on the invasive potential of colorectal cancer cells. Attached cells in the lower section were stained with hematoxylin and eosin and counted using light microscopy (magnification, x200). Control, cells without transfection; mock, cells transfected with mock vector; NPRL2, cells transfected with NPRL2-overexpression vector. NPRL2, nitrogen permease regulator-like-2; GFP, green fluorescent protein.

Mechanisms by which NPRL2 exerts its function

As shown in Fig. 6, the results of the western blotting demonstrated that the protein expression of p-AKT was significantly reduced in the NPRL2-transfected group, compared with the mock-transfected and control group in the HCT116 and HT29 cells (P<0.05), which indicated that NPRL2 may exert its function via inhibition of the Akt-mediated signaling pathway. As expected, the overexpression of NPRL2 induced a marked increase in the expression of caspase 3 and decrease in the expression of Bcl2 in the two cell lines (P<0.05), however, the expression of Bax was not affected (P>0.05).

Figure 6

Mechanism of function of NPRL2 on protein expression. Protein expression levels of p-AKT and the caspase-3, Bcl2 and Bax apoptosis-associated genes were determined using western blotting. Control, cells without transfection; mock, cells transfected with mock vector; NPRL2, cells transfected with NPRL2-overexpression vector. NPRL2, nitrogen permease regulator-like-2; p-, phosphorlyated; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Discussion

Allelic loss of the chromosome region 3p, particularly 3p21.3, has been observed as a frequent and early event in the development of several types of cancer, suggesting the presence of multiple TSGs in these regions (12,13). The 630 kb region contains ≥25 genes, whereas only 9 genes are located in or on the border of the 120 kb 3p21.3 region (4). This group of potential TSGs includes CACNA2D2, PL6, 101F6, FUS1, BLU, RASSF1 (RASSF1A and RASSF1C), HYAL1, HYAL2 and NPRL2. Extensive losses of protein expression or alterations in gene products have also been found in these genes in lung cancer and other types of primary cancer in humans. This suggests that similar mechanisms, including chromosome instability, aneuploidy, promoter methylation, haploinsufficiency and altered RNA splicing, and defects in transcriptional, translational and post-translational processes, which are frequently found in the 3p region and in 3p21.3 genes, may be important in the ultimate inactivation of these genes (4,14,15).

NPRL2 is 1351 bp long and encodes a protein of 380 amino acid residues, although the exact mechanism involved in the inactivation of NPRL2 remains to be elucidated, dysfunctional alterations of the NPRL2 gene and its products have been observed in various types of cancers and cancer cell lines in humans, including lung cancer, breast cancer, osteosarcoma and hepatocellular carcinoma (4–8). In CRC, Yogurtcu et al (9) investigated the mRNA expression of NPRL2 in 55 colon tumor and matched normal tissue samples using reverse transcription-quantitative polymerase chain reaction analysis. The expression of NPRL2 was significantly decreased in 45% of the patients. Lower expression levels of NPRL2 were significantly more frequently in poorly-differentiated tumor samples, compared with highly or moderately-differentiated tumor samples. Thus, decreased expression of NPRL2 has been hypothesized to contribute to the progression of CRC. The role of NPRL2 in the pathogenesis of CRC is further supported by a previous study, which investigated 62 patients with CRC, 38 patients with colorectal adenoma and 51 normal controls (10). The data revealed that the mRNA and protein expression levels of NPRL2 in the CRC samples was significantly lower than those in the adenoma or normal colorectal tissues. The mRNA expression levels of NPRL2 detected in the tumors were correlated with tumor stage and expression levels in the blood. Receiver operating characteristic analysis revealed that the expression of NPRL2 in the blood distinguishes colorectal adenomas and CRCs from normal controls. Furthermore, the mRNA expression levels of NPRL2 in the CRC tumor tissues and peripheral blood correlate with the progression of CRC. These results indicated that mRNA blood levels of NPRL2 may be a potentially useful marker for the detection of early stage adenomas and CRC. In addition, the expression of NPRL2 is negatively associated with the survival rate of patients with osteosarcoma (7) and HCC (8), indicating its value as an independent prognostic marker.

In the present study, lentiviral vector-mediated overexpression of NPRL2 was observed to inhibit growth, induce cell cycle G1 phase arrest, promote apoptosis and inhibit invasion in the HCT116 and HT29 human CRC cell lines in vitro. The functional investigation further confirmed the tumor suppressor role of NPRL2 in the pathogenesis of CRC. A previous study revealed that the reactivation of wild-type NPRL2, via recombinant adenoviral vector-mediated transfer, in lung cancer cells with abnormal 3p21.3 genes, inhibited tumor cell growth by inducing apoptosis and altering cell kinetics in vitro. In addition primary tumor growth was significantly suppressed and tumor progression and metastasis were inhibited following either intratumoral injection or systemic administration of a protamine-complexed recombinant adenoviral vector of NPRL2 in various human lung cancer mouse models (16). Similarly, a NPRL2-mediated tumor suppression effect has also been demonstrated in NPRL2-deficient KRC/Y renal cell carcinoma cells, U2020 small-cell lung carcinoma cells and A549 non-small-cell lung cancer cells, following the induction of the expression of NPRL2 at a physiological level (5).

However, the mechanism underlying NPRL2-mediated tumor suppressive activity remains to be elucidated. The effects of cisplatin are mediated through high levels of DNA damage, leading to programmed cell death or cell cycle arrest (17). Previous studies have demonstrated that re-expression of NPRL2 in NPRL2-negative and cisplatin-resistant cells significantly resensitizes the response of these cells to cisplatin treatment, as evidenced by reduced cell viability and increased apoptosis in vitro and in vivo, which suggests that NPRL2 is involved in DNA mismatch repair, cell cycle checkpoint signaling and regulation of the apoptotic pathway (18,19). 3-phosphoinositide-dependent protein kinase-1 (PDK1) is a key regulator of cell proliferation and survival signal transduction. PDK1 is known to be constitutively active and is further activated by Src-mediated phosphorylation. Kurata et al (20) performed Escherichia coli-based two-hybrid screening and revealed that NPRL2 forms a complex with PDK1 and suppresses Src-dependent tyrosine phosphorylation and activation of PDK1 in cells. In the present study, the protein expression of p-AKT was significantly reduced in the NPRL2-transfected cells, which indicated that NPRL2 may exert its function by inhibiting the Akt-mediated signaling pathway. Constitutively active AKT has also been found in a variety of types of cancer in humans (21). Furthermore, the present study also demonstrated that the overexpression of NPRL2 induced a marked increase in the expression of caspase 3 and a decrease in the expression of Bcl2 in the CRC cell lines, which confirmed the role of NPRL2 in the regulation of the apoptotic pathway.

In conclusion, the present study demonstrated that NPRL2 acts as a functional tumor suppressor in CRC cell lines, however, the mechanisms involved require further investigation.