The authors have declared that no competing interests exist.
Conceived and designed the experiments: XBZ ZC. Performed the experiments: MM MZ LW. Analyzed the data: LFL MZ. Contributed reagents/materials/analysis tools: LQ LFL XBZ ZC. Wrote the paper: KZ LW.
To investigate the effects of CCL21/CCR7 on the proliferation, migration, and invasion of T24 cells and the possible associated mechanisms: expression of MMP-2 and MMP-9, and regulation of BCL-2 and BAX proteins.
T24 cells received corresponding treatments including vehicle control, antibody (20ng/mL CCR7 antibody and 50 ng/ml CCL21), and 50, 100, and 200 ng/ml CCL21. Proliferation was evaluated by MTT assay; cell migration and invasion were assayed using a transwell chamber. Cell apoptosis was induced by Adriamycin (ADM). The rate of cell apoptosis was examined by flow cytometry using annexin V-FITC/PI staining. Western-blot was used to analyze MMP-2 and MMP-9 and BCL-2 and BAX proteins.
CCL21 promoted T24 cell proliferation in concentration-dependent manner with that 200 ng/mL induced the largest amount of proliferation. Significant differences of cell migration were found between CCL21treatment groups and the control group in both the migration and invasion studies (P < 0.001 for all). The expressions of MMP-2 and MMP-9 proteins were significantly increased after CCL21 treatment (p < 0.05 for all). Protein expression of Bcl-21 follows an ascending trend while the expression of Bax follows a descending trend as the concentration of CCL21 increases. No difference was found between the control group and antibody group for all assessments.
CCL21/CCR7 promoted T24 cell proliferation and enhanced its migration and invasion via the increased expression of MMP-2 and MMP-9. CCL21/CCR7 had antiapoptotic activities on T24 cells via regulation of Bcl-2 and Bax proteins. CCL21/CCR7 may promote bladder cancer development and metastasis.
Bladder cancer is one of the most common types of adult cancer. In 2008 alone, 386,000 patients were diagnosed with bladder cancer which resulted in 150,200 deaths worldwide based on global statistics[
Physiologically, CCL21/CCR7 plays important roles in homing of immune cells, lymph-node homing and positioning, immunity and peripheral tolerance, development and function of T regulatory cells, and autoimmunity and lymphoid neogensis[
The purposes of the present study were to investigate the effects of CCL21/CCR7 on the proliferation, migration, and invasion of T24 cells and the possible associated mechanisms: expression of MMP-2 and MMP-9, and regulation of BCL-2 and BAX proteins.
This study obtained ethics approval from the ethics committee at Xiangya Hospital, Central South University, Changsha, Hunan Province, China.
CCL21 recombinant human protein was purchased from Perprotech (Rocky Hill, NJ, USA) and polyclonal rabbit anti-human CCR7 antibody was purchased from Wuhan Boster Biological Engineering Co., Ltd. (Wuhan, China). Protease inhibitor Phenylmethylsulfonyl fluoride (PMSF) was purchased from Roche Diagnostics (Indianapolis, IN, USA). MTT and Dimethyl sulfoxide (DMSO) was purchased from Hufeng Chemical Co., Ltd. (Shanghai, China). Annexin V-FITC apoptosis detection kit was purchased from Beyotime Biotechnology (Shanghai, China).
The human bladder cancer T24 cell line was purchased from the Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences (Shanghai, China). The cells were cultured at 37°C, 5% CO2, in medium of DMEM (GIBCO, USA) containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin.
T24 cells were treated for 48 hours prior to MTT assay, migration and invasion assays, and Western blot analysis and 24 hours prior to flow cytometry study. For each experiment, there were five groups consisting of Group 1 (control group) which was treated with serum free DMEM medium, Group 2 (antibody group) which first received pretreatment of 20 ng/mL of polyclonal rabbit anti-human CCR7 antibody for 4 h then 50 ng/ml of CCL21, Group 3 which received 50 ng/ml of CCL21, Group 4 which received 100 ng/ml of CCL21, and Group 5 which received 200 ng/ml of CCL21. The experiments of MTT assay, flow cytometry, and Western blot were repeated three times (n = 3), and cell migration and invasion were repeated four times (n = 4).
Proliferation of T24 cells was evaluated by MTT assay. In brief, cells were treated correspondingly in each group for 48 hours. Following treatment, the medium was removed and the cells were incubated with 5mg/mL of MTT solution (20μl). After incubation for 4 h at 37°C and 5% CO2, the supernatant was removed and formation of formazan was measured at 490 nm with Gel Documentation & Analysis System set (Liuyi Factory, Beijing, China).
Cell migration and invasion were assayed using a transwell chamber (EMD, Millipore, USA). For the invasion assay, a transwell chamber was placed into a 24-well plate and was coated with 30 μl Matrigel (Franklin Lakes, USA) and was incubated for one hour at 37°C. In both transwell assays, cells, after 48 hours' corresponding treatment, were trypsinized and seeded in chambers at the density of 8 × 104 cells per well, and 500 μl of serum free DMEM medium was added to the lower chamber. Migrated cells were fixed with 100% Matrigel (BD, Franklin Lakes, USA) methanol for 30 min after 24 hours, and non-migrated Cells were removed by cotton swabs. Finally, cells on the bottom surface of the membrane were stained with hematoxylin and eosin for 20 min. Optical microscope (200X, Olympus, Tokyo, Japan) was used to count the number of cells in five random fields of view; mean cell number was calculated for each group.
Cell apoptosis was induced by Adiamycin (ADM). The rate of apoptosis of T24 cells was examined by flow cytometry using annexin V-FITC/PI staining. Briefly, T24 cells were treated as above in each group for 24 h; cells were then incubated with ADM (0.1mg/ml) for additional 48 h. Then cells were harvested, washed and resuspended in PBS. Apoptotic cell death was measured by double staining annexin V-FITC and PI using the annexin V-FITC apoptosis detection kit (Beyotime Biotechnology, Shanghai, China) as per the manufacturer’s instructions. Flow cytometric analysis was performed immediately after staining. Data acquisition and analysis were performed by flow cytometry using the Cell Quest software.
T24 cells were placed into 75 mL culture vials and were incubated at 37°C for 24 h and then treated correspondingly in each group for 48h incubation. Suspensions were washed with cold PBS. The cells were then incubated in ice-cold RIPA buffer [1 M Tris (pH 7.4), 5 M NaCl, 0.5 M EDTA (pH 8.0), 10% SDS, 10% DOS, and 10% NP40] with fresh protease inhibitor PMSF over ice for 20 min. The cells were scraped and the lysate was collected in an Eppendorff tube and centrifugated at 10,000 rpm at 4°C for 10 min, and the supernatants were collected, aliquoted, and stored at -20°C for future use.
Dilutions of BCL-2/BAX (12%) and MMP-2/MMP-9 (7.5%) were used for the study. Proteins (20 μg) were loaded in 5–15% SDS-PAGE gels and transferred onto a nitrocellulose membrane (Amersham Biosciences, USA). The membranes were soaked in blocking buffer (5% skimmed milk) under room temperature for 2 h. The blots were washed with PBS (with 0.1% BSA). To probe for MMP-2, MMP-9, BCL-2, Bax, and β-actin, the membranes were incubated 2h under room temperature with relevant antibodies, followed by appropriate HRP conjugated secondary antibodies and ECL detection. Gel Documentation & Analysis System set (Liuyi Factory, Beijing, China) was used for picture capture and analysis of Optical density (OD) values (490nm).
Statistical analysis was performed using the SPSS 18.0 statistical software. Quantitative data were expressed as the mean ± SD. One-way analysis of variance with Fishers LSD test was used for group comparisons. The significance level was set at p-value less than 0.05.
The effects of CCL21/CCR7 on T24 cells as represented by OD values are presented in
Groups | OD value | P-values |
---|---|---|
Control (DMEM medium) | 0.211 ± 0.013 | — |
Anti-CCR7 Ab + CCL21 50 ng/mL | 0.216 ± 0.011 | P > 0.05 |
50ng/mL Group | 0.248 ± 0.006 | P < 0.05 |
CCL21 100 ng/mL | 0.290 ± 0.004 | P < 0.01 |
CCL21 200 ng/mL | 0.341 ± 0.012 | P < 0.001 |
Note: The OD values present Mean ± SD from 3 independent experiments.
The results of CCL21/CCR7 on the migration and invasion of T24 cells are presented in
The assay was performed using a transwell chamber coated with 30 μl of Matrigel mixture solution with five different groups consisting of vehicle control, CCR7 antibody 20 ng/ml plus CCL21 50 ng/ml, CCL21 50 ng/ml, CCL21 100 ng/ml, and CCL21 200 ng/ml. The data present Mean ± SD from four independent experiments.
Western blot results are presented in
Lane 1: T24 cells treated with vehicle control; Lane 2: T24 cells treated with CCR7 antibody 20 ng/ml plus CCL21 50 ng/ml; Lane 3: T24 cells treated with 50 ng/ml CCL21; Lane 4: T24 cells treated with 100 ng/ml CCL21; and Lane 5: T24 cells treated with 200 ng/ml CCL21. Cell lysates were prepared and run on 5–15% SDS-PAGE gels following by a Western blot analysis. Beta-actin was used as a loading control. The blots shown are representative of three independent experiments.
Groups | MMP-2 | MMP-9 |
---|---|---|
Control | 0.26 ± 0.03 | 0.17 ± 0.04 |
Anti-CCR7 Ab + CCL21 50 ng/ml | 0.32 ± 0.05 | 0.20 ± 0.06 |
CCL21 50 ng/ml | 0.43 ± 0.05 | 0.33 ± 0.05 |
CCL21 100 ng/ml | 0.67 ± 0.07 | 0.41 ± 0.06 |
CCL21 200 ng/ml | 0.80 ± 0.10 | 0.65 ± 0.08 |
Note: The OD values present Mean ± SD from 3 independent experiments.
Effects of CCL21/CCR7 on the expressions of Bcl-2 and Bax by Western blot are presented in
T24 cells were treated with vehicle control, CCR7 antibody 20 ng/ml plus CCL21 50 ng/ml, CCL21 50 ng/ml, CCL21 100 ng/ml, and CCL21 200 ng/ml. The values (OD) present Mean ± SD from three independent experiments.
Early and late apoptosis rates and total cell death rates after treatments are presented in
Groups | Early apoptosis (%) | Late apoptosis/necrosis (%) | Total cell death (%) | ||||
---|---|---|---|---|---|---|---|
Control | 26.4 ± 5.0 | 37.5 ± 2.4 | 63.9 ± 7.1 | ||||
Anti-CCR7 Ab 50 ng/ml + CCL21 50 ng/ml | 25.2 ± 3.8 | 35.6 ± 5.5 | 60.8 ± 9.3 | ||||
CCL21 50 ng/ml | 19.6 ± 2.7 | 16.5 ± 2.1 | 36.1 ± 1.0 | ||||
CCL21 100 ng/ml | 14.9 ± 1.8 | 17.8 ± 1.4 | 32.7 ± 1.5 | ||||
CCL21 200 ng/ml | 17.7 ± 1.4 | 16.4 ± 3.3 | 34.1 ± 2.4 |
Note: The values present Mean ± SD from 3 independent experiments.
The potential of cancer cells to metastasize depends on its interactions with homeostatic factors including chemokines which not only promote cancer cell growth and survival but also migration and invasion or metastasis. Previous studies have demonstrated that chemokines are involved in the progression and metastasis of various cancers[
Chemokine concentration may be at least in part responsible for cancer development and metastasis[
The interaction of chemokines and their receptors serve as the foundation for tumor cell motility and migration via triggering intracellular actin polymerization to create pseudopodia[
Molecules like chemokine CCL21 are involved in the metastasis of cancers not only via mediating the migration and invasion of cancer cells into tissues but also providing the required supportive microenvironments. In vivo, the migration of cancer cells is often slowed with lots of resistances, such as basement membrane and surrounding connective tissue[
Using radiolabled techniques, researchers have found only a tiny portion of radiolabled cancer cells in microcirculations has the potential to metastasize[
The result of the flow cytometry can be divided into four quadrants: the lower left quadrant with normal alive cells [Annexin-V (−), PI (−)], the upper right quadrant [Annexin-V (+), PI (+)] with late apoptotic or necrotic cells, the lower right quadrant [Annexin-V (+), PI (−)] with early apoptotic cells, and the upper left quadrant [Annexin-V (−), PI (+)] with small portions of cell debris due to mechanical injury and apoptosis. Thus, the analysis of apoptosis mainly depends on early apoptosis (lower right quadrant) and total cell death (upper right + lower right quadrants) in the present study. Our results demonstrated significant decrease of apoptosis and cell death with CCL21 treatment compared to the control (p < 0.01); however, no difference between CCR7 antibody plus CCL21 treatment and the control was found (p > 0.05). The results indicate that CCR7 antibody could antagonize the effect of CCL21 on apoptosis and cell death and thus confirm the antiapoptotic function of CCL21/CCR7. However, no difference of apoptosis was detected among the three treatment groups (p > 0.05). The possible cause could be that CCL21 at 50 ng/mL reached saturate condition thus higher concentrations could not further increase it effect.
Bcl-2 family is found to be closely related to cell apoptosis[
CCL21/CCR7 promoted T24 cell proliferation in a concentration dependent pattern and enhanced its migration and invasion; these effects may be related to the increased expression of MMP-2 and MMP-9. CCL21/CCR7 had antiapoptotic activities on T24 cells; these functions may be related to the regulation of Bcl-2 and Bax proteins. Our results indicate that CCL21/CCR7 may promote bladder cancer development and metastasis.