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N-Myc downstream-regulated gene 2 is down-regulated in some carcinomas. The aim of this study was to investigate its expression in bladder cancer tissues and several bladder cancer cell lines, in order to elucidate further its clinical and pathological significance. 97 bladder carcinoma and 15 normal bladder tissue sections were analyzed retrospectively with immunohistochemistry. The human bladder cancer cell line T24 was infected with LEN-NDRG2 or LEN-LacZ. The effects of NDRG2 overexpression on T24 cells and T24 nude mouse xenografts were measured via cell growth curves, tumor growth curves, flow cytometry (FCM), western blot and Transwell. NDRG2 was highly expressed in normal bladder tissue, but absent or lowly expressed in cacinomatous tissues (χ2=8.761, p < 0.01). The NDRG2 level was negatively correlated with increasing tumor grade and pathologic stage(r=-0.248, p < 0.05), as well as increased c-myc level (r=-0.454,p< 0.001) . It was positively correlated with p53 (r=0.669, p< 0.001). The expression of NDRG2 was low in the three BC cell lines. T24 cells infected with LEN-NDRG2 showed inhibition of proliferation both in vitro and in vivo, and NDRG2 overexpression can inhibit tumor growth and invasion in vitro.
The incidence of bladder cancer is increasing. An estimated 386,300 new cases and 150,200 deaths from bladder cancer occurred in 2008 worldwide . In men, bladder cancer is the fourth most common cancer only after prostate, lung, and colorectal cancers, accounting for 7% of all cancer cases in USA . In more than 70% of the cases, the diagnosis is made at an early stage of disease (stages Ta and T1) [?]. Despite have done adequate control, the five-year overall survival rate for pathologic T2 disease is 52-77%, T3 disease 40-64%, and T4 or node-positive disease 26-44% . There are many therapeutic on this disease; however, the survival rates have not changed during the last 20 years. Therefore, new antioncogene agent, which improve the survival of patients, are most necessary.
The N-myc downstream regulate gene 2 (NDRG2) belongs to the NDRG family, which is comprised of 4 members: NDRG1, NDRG2, NDRG3 and NDRG4. The sequence homology within the human NDRG family is 57-65% and the gene family has been investigated in human nervous system disorders and cancer . As a gene that is regulated downstream of Myc, NDRG2 expression has been shown to be reduced in many types of carcinomas, including liver cancer, pancreatic cancer, thyroid cancer, meningioma and prostate cancer [5-11]. Those studies suggest that NDRG2 might play an important role in the morbidity of carcinomas. The NDRG2 has been confirmed to be involved with cell growth and differentiation, meanwhile, NDRG2 expression in high-grade gliomas was positively correlated with survival [12, 13].
There has not been a functional characterization of a potential role of NDRG2 in bladder cancer. Therefore, the aim of the current study was to investigate the role of NDRG2 in human bladder cancer. First, we examined the expression of NDRG2 in human bladder carcinoma tissues and normal bladder tissues by immunochemistry, finding the BC tissues to have lower expression levels than normal tissues. Next, we used bladder cancer cell lines as models to evaluate the effects of NDRG2 on tumor growth, differentiation and invasion in vitro and in vivo. Our results suggest a potential antioncogenic role of NDRG2 in bladder cancer.
2. Materials and methods
2.1. Clinical samples
A total of 112 formalin-fixed and paraffin-embedded blocks of bladder carcinoma and normal tissues were randomly collected from the Department of Urologic Surgery, Xijing Hospital, FMMU (Xi'an, China) between 2008 and 2011 (mean age 63.4 years, age range 21-81 years). Tis sample was comprised of 15 normal bladder tissues, 20 bladder papilloma, 38 low-level bladder cancer and 39 high-level bladder cancer samples. All samples were obtained from patients who gave informed consent to use excess pathological specimens for research purposes.
Mouse anti-human NDRG2 monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The c-Myc and p53 monoclonal antibody (mouse anti-human) and an immunohistochemistry kit were purchased from the Boster Company (Wuhan, China). The immunohistochemistry staining was performed according to the manufacturer's instructions. The following preparations were made from each tissue block: a slide incubated with NDRG2 antibody, a slide incubated with p53 antibody, and a slide incubated with p53 antibody.
Both the intensity and the extent of immunological staining were analyzed semi-quantitatively. The ratio of positive cells per specimen was evaluated quantitatively and scored as 0 for staining of <5% of the cells examined, 1 for staining of 6-25%, 2 for staining of 26-50%, 3 for staining of 51-75%, and 4 for staining >75%. The staining intensity was scored similarly, 0 used for negative staining, 1 for weakly positive, 2 for moderately positive and 3 for strongly positive. The scores for the percentage of positive cells and for the staining intensity were multiplied to generate an immunoreactive score for each specimen. The products was calculated such that a final score of 0 indicated no expression, 1-4 indicated weak expression, 5-8 indicated moderate expression and 9-12 indicated strong expression. Each sample was examined separately and scored by two pathologists. Cases with discrepancies in the scores were discussed to reach a consensus. Imaging was performed via light microscopy with the aid of SPOT Advanced Software (Olympus, Nagano, Japan).
2.3. Cell culture
T24, 5637 and BIU-87 are the most frequently used cell lines in the study of BC, and we chose normal human bladder cells (SV-HUC-1) as the control. After resuscitation, the cells were maintained in phenol red-free RPMI1640 (Gibco) containing 100 ml/L fetal bovine serum (Haoyang Tianjin, China) at 37°C in a humidified atmosphere with 5% CO2 concentration. When the cells grew to approximately 80% confluence in 25 cm2 plastic culture flasks, we collected them through trypsinization.
2.4. Real-time PCR and Western blotting analysis
Total RNA was extracted from cells using Trizol reagent (TaKaRa,Japan) and reverse transcribed using M-MLV Reverse Transcriptase (Fermentas) following the manufacturer's protocol. The primers used were as follows: for GAPDH, 5′-AGGTCCACCACTGACACGTT-3′ and 5′-GCCTCAAGATCATCAGCAAT-3′; for NDRG2, 5′-GCCCAGCGATCCTTACCTACC-3′ and 5′-GGCTGCCCAATCCATCCAACC-3′. The amplification program consisted of polymerase activation at 95°C for 30 seconds and 40 cycles of denaturation at 95°C for 15 seconds, annealing and extension at 59°C for 30 seconds. All experiments were repeated at least three times.
Western blot was performed as follows. After extracting all of the protein from each of the cell lines with lysis buffer, the protein samples (50μg) were separated by SDS-PAGE at 10% concentration. Next, gels were electroblotted onto nitrocellulose membranes (Amersham, St. Giles, UK). After that, the blots were incubated in milk containing mouse anti-human NDRG2 monoclonal antibody (1:500 dilution) for 1 h at room temperature and overnight at 4°C. GAPDH protein (36 kDa) detection was used as an internal control. After three washes for 15 min in TBS-T, the blots were incubated at room temperature for 2 h with horseradish-peroxidase (HRP)-conjugated anti-mouse or anti-rabbit secondary antibody (dilution 1: 2000, Santa Cruz Biotechnology). The enhanced chemiluminescence (ECL) system detection solutions (Pierce, NJ) were then applied. Scanned images were quantified using Kodak Digital Science ID software (Kodak, NY).
2.5. Lentivirus infection
To produce lentivirus, 293T cells were co-transfected with pLEN-GFP-NDRG2 or pLEN-GFP-Lacz and plasmids which were amplified in E. coli DH5, purified using a QIAGEN Plasmid Maxi Kit (Qiagen, Valencia, CA), and transfected into 70% confluent 293T cells using lipofectamine 2000 (Invitrogen). Lentiviral particles were harvested from the supernatant 72 hours after transfection and purified by ultracentrifugation . These particles are hereafter referred to as LEN-NDRG2, and LEN-Lacz (negative control). Stably infected T24 cells were selected using blasticidin, by counting green fluorescent protein (GFP)-positive cells under fluorescence microscopy (Olympus, Japan), applied at the minimum concentration of blasticidin(using the serial dilution method) required to kill uninfected T24 cells.The cell line was divided into the following 3 experimental groups: the CON group (non-infected cells), the Lacz group (LEN-Lacz-infected cells), and the NDRG2 group (LEN-NDRG2-infected cells).
Both Real-time PCR and Western blot confirmed that we successfully infected T24 cells with the lentiviral and over-express NDRG2 protein properly after infection. We also ascertained the differential expression of proteins that correlated with cell cycle stage, apoptosis and cell migration and invasion (cell cycle protein: cyclinD1, CDK4; cell apoptosis protein: p16; cell migration and invasion protein: nm23-H1, MMP-2, MMP-9).
2.6. Cell growth inhibition tests in vitro
The tests included MTT assays, Colony formation assays, flow cytometry assays (FCM) (BD Company, NJ). Each one was applied to each of the three groups (PBS-treated cells, the blank control; LacZ, the negative control; and NDRG2, the experimental group). The experiments were repeated five times.
(1) MTT assay: All the cells, including those infected, were grown in exponential phase and detached by trypsin treatment. Viable cells (2000 cells/ml) were inoculated into 96-well plates and every group had six reduplicative wells. At different time points, MTT reagent was added 20 μl/well (5mg/ml) and incubated at 37 °C for 4 h. The reaction was stopped by the addition of 150 μl DMSO followed by shaking for 10 min. Absorbance (A) values were measured with an autokinetic enzyme scaling meter (Bio-Rad, CA) at 490 nm wavelength. Cell growth curves then were then drawn based on the average A values.
(2) Colony formation assay: Cells were seeded into six -well plates (200 cells /well) (in three duplicate wells) and cultured at 37°C in 5% CO2. After two weeks, the cells were fixed with methanol for 20 min and then stained with Giemsa for 20 min. ddH2O was used to wash the cells three times to obtain a clean background. The number of colonies and the cell number in each colony were counted and statistically analyzed.
(3) FCM: T24 cells (5 x 10 5 cells/ well) were plated in six-well culture dishes with the infected LEN-NDRG2 lentivirus. After 48 h and 72 h, cells were harvested, centrifuged at low speed and fixed in 70% ethanol. After overnight incubation at 4°C, cells were stained with 50 μ g/ml propidiumiodide in the presence of RNAseA (10 μg/ ml) and 0.1% Triton X-100, then measured with a flow cytometer.
2.7. Migration and invasion assays
Invasion assay with a Matrigel-coated membrane and migration assay with a Matrigel-uncoated membrane were performed using a 24-well chamber system (BD Biosciences, Bedford, MA), according to the manufacturer's instructions. The cells were trypsinized and seeded in the upper chamber at 2.5x104 cells/ well in serum-free medium. Culture medium supplemented with 30% FBS (fetal bovine serum) (used as a chemo-attractant) was placed in the bottom well. Incubation was carried out for 24 h at 37°C in humidified air with a 5% CO2 atmosphere. The cells were allowed to migrate through a porous, Matrigel-coated or uncoated membrane (BD Biosciences). After the incubation, the chambers were removed, and invading cells on the bottom side of the membrane were fixed with methanol and stained with Kristallviolet. The number of invading cells or migrating cells were determined by counting five high-power fields (400-) on each membrane and calculated as the mean number of cells per field.
2.8. Growth inhibition assays in vivo
Male BALB/c nude mice were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences, Shanghai, China. T24 cells were harvested and resuspended in sterile PBS. T24 cells (1 x 107) in 0.2 ml were injected subcutaneously into the left flanks of the 6-week-old nude mice. The mice were randomly divided into two groups (LEN-NDRG2, LEN-LacZ, n=5 per group). When the mean size of tumors reached 300 mm3 (as calculated by the equation: V [mm3] = ab2 /2) in vivo, the mice were sacrificed by cervical dislocation and tumor specimens were taken, photographed, measured and weighed. The expression levels of NDRG2 protein in the inoculated tumors were detected using western blot. We also measured the expression of our other marker proteins again (cell cycle protein: cyclinD1, CDK4; cell apoptosis protein: p16; cell migration and invasion protein: nm23-H1, MMP-2, MMP-9).
2.9. Statistical analysis
Statistical analyses were performed using SPSS17.0 software (SPSS company, IN). All data are represented as the mean ± standard error of at least three independent experiments. For immunohistochemistry results, differences across groups were validated using Chi-square tests and Pearson correlation. Analysis of variance (ANOVA) , Student-Newman-Keuls (SNK) tests and non-parametric test were performed to determine whether there were differences among the results of assays in vitro and in vivo. A p-value less than 0.05 was considered statistically significant.
3.1. Differential expression of NDRG2 in normal bladder tissues and bladder carcinoma tissues
As shown in Table 1, the rate of NDRG2 positive expression in normal bladder tissues specimens was 80 % (12/15), which was significantly higher than that in bladder carcinoma tissues 39.17% (38/97) (χ2= 8.761, p<0.01). NDRG2 expression was not correlated with gender or age, but was closely correlated with pathologic stage (r=-0.248, p<0.05) (Table 2). That is to mean, higher degree of malignancy were associated with decreasing percentages and levels of NDRG2 expression. Immunohistochemistry showed NDRG2 protein in the cytoplasm of normal tissues and bladder carcinoma (Fig.1A). The expression of NDRG2 in bladder carcinoma was negatively correlated with c-Myc in bladder carcinoma (r=-0.454, p<0.001) (Figs. 2).
3.2. Differential expression of NDRG2 in normal and bladder cancer cell lines
Both Real-time PCR and western blot results showed that all three BC cell lines expressed lower levels of NDRG2 protein and mRNA than the normal human bladder cells (SV-HUC-1), and the T24 had the lowest level among the three BC cell lines (Fig. 3A and B).
3.3. Lentivirus infection and cell growth inhibition tests
Fluorescence microscopy was used to determine that the efficiency of infection was more than 90% (Fig.?). Real-time PCR analysis showed that the mRNA expression of NDRG2 in T24 cells in the LEN-NDRG2 group was significantly increased; the mRNA level of Med19 was 95% higher in the LEN-NDRG2 group than in the LEN-Lacz and CON groups (P<0.05, Fig.). Western blot analysis showed that the NDRG2 protein in the LEN-NDRG2 group was increased compared with that in the NC and CON groups (Fig.4C). The LEN-NDRG2 could cause overexpression of NDRG2 in T24 cells. The cell growth curves and colony formation showed that overexpression of NDRG2 could suppress the growth of T24 cells more than that of the other two groups. In the cell growth curves assays, the inhibition began at the third day and became increasingly powerful thereafter (F≥44.58, p≤ 0.001) ( Fig. 4B and 4C). The FCM results showed that T24 cells infected with LEN-NDRG2 lentivirus were more easily arrested in G0 /G1 cycle than the negative control and LEN-LacZ groups (48 h: F = 41.92, p < 0.001; 72 h: F = 24.11, p < 0.001) (Fig. 5 and Table. 3). The western blot results showed that overexpression of NDRG2 could downregulate cyclinD1 and CDK4, while upregulate p16 in T24 cells (Fig. 6A and B).
3.4. NDRG2 gene transfer inhibits the invasion and migration of human bladder cancer cells in vitro
Transwell migration assays and Matrigel-coated transwell invasion assays were performed with T24 cells. The representative micrographs were taken from the lower surface of the transwell filter, and cells that migrated or invaded were stained with Kristallviolet. As shown in Fig. 7A, the invasive potential, which is determined by the cells' ability to invade a Matrigel barrier, was also considerably suppressed in NDRG2-overexpressing cells (P<0.01). Furthermore, forced expression of NDRG2 in T24 cells significantly suppressed their migration through the transwell (P < 0.01; Fig. 7B).
3.5. LEN-NDRG2 treatment suppresses expression of nm23-H1, MMP-2 and MMP-9 in T24 cells
To understand the molecular basis of the NDRG2 antagonism of T24 cells metastasis, we analyzed the effect of NDRG2 on the expression of the genes including nm23-H1, MMP-2 and MMP-9 which play an important role in bladder cancer metastasis. We examined nm23-H1, MMP-2 and MMP-9 expression by gel blot analysis (Fig. 7C and D) which showed that LEN-NDRG2 significantly reduced nm23-H1 expression and slightly reduced MMP-2 and MMP-9 expression compared to LEN-LacZ infected T24 cells.
3.6. Growth inhibition assays in vivo
Tumors were allowed to grow for 35 days after inoculation with T24 cells (median size = 300 mm3 in each group). Five mice in each group survived after the injections with lentivirus. Tumor growth curves showed that NDRG2-overexpression could suppress the growth of the induced tumors (Fig. 8A). The discrepancy had statistical significance from 3 weeks onwards (F≥31.65, p<0.001) (Fig. 8B). The weight of tumorsinduced by LEN-NDRG2 group was significantly lighter than those of LEN-LacZ tumors (F≥39.82, p<0.001) (Fig. 8D). The ratio of tumor weight to mouse weight indicated that the physical condition of the mice in LEN-NDRG2 group was much better than that of the mice in LEN-LacZ group (F = 39.81, p < 0.001) (Fig. 8C). When we anatomized the mice, we found ipsilateral lymph node metastases (but no other organ involvement) in the LEN-LacZ group, however, in the LEN-NDRG2 group we did not find any metastatic lesions(Fig. 8A). Western blot showed that LEN-NDRG2 lentivirus could upregulate expression of NDRG2 protein in the tumors, and that overexpression of NDRG2 could downregulate cyclinD1, CDK4, p16, MMP-2, MMP-9, and nm23-H1 both in vivo and in vitro (Fig. 9A and B).
The high recurrence of bladder cancer and significant metastatic potential pose great challeges to care and prognosis, approximately 75% of all bladder carcinomas recur within the first 5 years . Therefore, facilitating early diagnosis and finding new therapies are crucial goals in the study of bladder cancer. NDRG2 is a candidate tumor suppressor gene, and a series of studies have suggested that it play an important role in cell proliferation, apoptosis and metastasis [15, 16]. Some of the results of our study reinforce this understanding.
We demonstrated that NDRG2 plays an important role in bladder cancer that is similar to its role in other malignant tumors. Immunohistochemistry results showed that the positive expression percentage of NDRG2 in bladder carcinoma tissues was significantly lower than in normal bladder tissues. The expression levels of NDRG2 protein decreased as the degree of bladder carcinoma malignancy increased. We also found that the expression of NDRG2 was negatively correlated with c-Myc and positively correlated with p53 in bladder carcinomas, just as it is in other tumor cells. Meanwhile, the NDRG2 expression levels in bladder cancer cell lines (T24, 5637 and BIU-87) were lower than normal bladder cell line (SV-HUC-1).
Lentiviruses are particularly well-suited for gene therapy because they can steady integrate into the host genome to provide long expression of target gene, furthermore, lentiviruses have been rendered progressively safer by the development of split plasmid systems for vector production to prevent generation of replication-competent virus .Overexpression of NDRG2 was able to inhibit the proliferation of T24 cells, to promote their apoptosis both in vitro and in vivo, and to suppress the invasion and migration of human bladder cancer cells in vitro. Overexpression of NDRG2 affected the expression of several proteins (cyclinD1, CDK4 and p16) that are important in cell cycle regulation and apoptosis, and overall, NDRG2-overexpression can improve the physical condition of mice with inoculated tumors.
The expression of MMP-2 and MMP-9 is upregulated in bladder cancer cells predisposed to invasion [17, 18], and the expression of nm23-H1 play an important role in the suppression of early tumorigenesis in the human bladder . Thus, these genes may be important in elucidating the molecular mechanism of bladder cancer invasion and migration. In this study, western blot assay showed that LEN-NDRG2 in T24 cells was associated with significant reduction in nm23-H1 andMMP-9 expression and slightly reduced MMP-2 expression. This suggests that NDRG2-overexpression may regulate MMP-2, MMP-9 and nm23-H1 activity and inhibit the invasion ability of metastatic bladder cancer cells.
In conclusion, we demonstrated the antioncogenic role of NDRG2 in the development and invasion of bladder cancer. Since NDRG2 is implicated in many aspects of tumor progression, including cell growth, cell cycle regulation, invasion and migration, it represents a promising therapeutic target for bladder cancer. However, whether the down-regulation of NDRG2 in bladder cancer is a cause or a consequenceof carcinogenesis still remains unclear. Further studies are needed to investigate how NDRG2 is involved in the progression from normal bladder tissue to bladder cancer tissue. Still, our findings not only provide a molecular understanding of the role of NDRG2 in bladder cancer, but also suggest a novel therapeutic approach for bladder cancer.
Conflict of interest
This study was supported by grants from the Nature Science Foundation of China (Project No. 31070681).