Table 1.
Primer sequences used for real-time RT-PCR.
Fig 1.
Establishment of CD10-A375 melanoma cells and gene expression profiles.
(A) Stable transfection of CD10-A375 cells was confirmed by flow cytometry. After selection with G418, CD10-A375 or mock-A375 cells were examined for CD10 protein expression with PE-conjugated anti-hCD10 antibody. CD10-A375 exhibited marked surface expression of CD10 (gray shaded area), whereas mock-A375 did not (solid gray line). Isotype IgG control is shown with a gray dashed line. (B) Western blot analysis of CD10 and GAPDH in the wild type- and transfected- A375 cells. Successful transfection of CD10 was also confirmed in CD10-A375 cells by Western blot analysis. (C and D) Microarray analysis of gene expression in CD10-A375 vs. mock-A375 melanoma cells. (C) Heat map generated using MeV4.6 from the 1,247 probes of genes significantly differentially expressed between CD10-A375 and mock-A375 (n = 4 per group). A red color represents a higher-than-average standardized expression value, whereas a green color represents a lower-than-average one. Examples of differentially expressed genes are listed and sorted into major known functional categories in Table 2. (D) Heat map of the selected genes as shown in Table 2. (E) qRT–PCR validation analysis of representative genes that were significantly upregulated in CD10-A375 (n = 3). Each value was normalized for ACTB (β-actin), and the results are expressed as fold change over mock-A375. Primers used in the assay are shown in Table 2. *P < 0.05, **P < 0.01, ***P < 0.001; unpaired t test.
Table 2.
A list of differentially expressed genes in CD10-A375 cells compared with mock-A375 cells.
Fig 2.
CD10 promotes in vitro cell proliferation and in vivo tumor growth, but its tumorigenic effect can be suppressed by CD10 enzymatic inhibitors.
(A) CD10 expression promotes the proliferation of A375 melanoma cells in vitro. Growth curves of CD10-A375 (solid line) and mock-A375 (dotted line) cells grown in 5% FBS (n = 3 per group) show that CD10 expression increases cell growth. Cells were plated in triplicate in 96-well plates and incubated for 24, 48, 72, or 96 hours, and the number of viable cells was assessed, as described in Materials and Methods. Points, mean (n = 3); bars, SD. Cell proliferation rate was significantly higher in CD10-A375 (solid line) than in mock-A375 (dotted line). Representative data of three independent experiments are shown. (B) Effect of CD10 overexpression by A375 cells on tumor growth in a xenograft model. A total of 5 × 105 CD10-A375 or mock-A375 cells resuspended in 100 μl of PBS were injected i.d. into the backs of BALB/C nude mice, and tumor growth was observed for about 28–35 days. Tumor diameters were measured and used to assess tumor volume, as described in Materials and Methods. Points, mean (n = 6); bars, SE. Each group consisted of six mice. CD10-A375 (solid line) manifested significantly higher tumorigenicity in vivo than mock-A375. Representative data of three independent experiments are shown. (C) Representative example of nude mice 30 days after tumor implantation, showing mice inoculated with CD10-A375 (right) or mock-A375 (left). It is noteworthy that mice injected with CD10-A375 tended to have ulceration on the tumor surface. (D) Immunohistochemical staining of a xenograft model for CD10. Upper, tumor from mock-A375-injected mouse; lower, tumor from CD10-A375-injected mouse. Staining is shown in red. Tumors from mice injected with CD10-A375 (lower) showed strong staining in tumor cells. Representative pictures of each group are shown. (E) Preventive effects of CD10 inhibitors on mouse tumor growth. Mice were injected i.d. with 5 × 105 viable CD10-A375 cells resuspended in 100 μl of PBS with or without CD10 inhibitors. Phosphoramidon (20 μg/mouse) or thiorphan (20 μg/mouse) was administered intraperitoneally before the tumor cell injection. Points, mean (n = 6); bars, SE. Each group consisted of six mice. Both inhibitors significantly and markedly inhibited mouse tumor growth. Representative data of three independent experiments are shown. *P < 0.05; **P < 0.01; ***P < 0.001 by two-way ANOVA.
Fig 3.
CD10 decreases cell migration and invasion.
Cell migration assay (A-D) and invasion assay (E) of the CD10-A375 or mock-A375 cells were performed in the absence (A, B) or presence (C, D) of mitomycin C (10 μl/ml). (A and C) Representative images showing the wound gap filled by CD10-A375 (right) or mock-A375 cells (left) cultured in the absence of mitomycin C for 87 hours (A) or in its presence at a concentration of 10 μl/ml for 24 hours (C). (B and D) Graphs showing the time course changes in the percentage of wound healing after 87 hours of incubation without mitomycin C (B) or after 24 hours of incubation with mitomycin C (D). CD10-A375 (white dot) displayed lower migratory capacity both in the absence and in the presence of mitomycin C, compared with mock-A375 (black dot). ***P < 0.001 by two-way ANOVA. Results are representative of at least three independent experiments, and values represent averages of three independent measurements along the wound. (E) A graphic result of cell invasion assay of CD10-A375 vs. mock-A375 cells. Cells were plated on polycarbonate membrane inserts coated with uniform layer of basement membrane in 24-well plates (Cell Biolabs) and incubated for 72 hours. The invading cells were stained and quantified as indicated in the graph at 560 nm.
Fig 4.
CD10 exhibits higher resistance to anticancer drugs.
(A-C) Histogram (upper) and dot plot (lower) showing the proportion of Annexin V- and Annexin V/PI-positive cells 24 hours after treatment with anticancer drugs. CD10-A375 or mock-A375 cells were treated with etoposide (B) or gemcitabine (C) overnight. Cell were harvested and stained with Annexin V and PI, and 10,000 events were counted by flow cytometry. Without any treatment (A), the number of Annexin V-positive apoptotic or Annexin V/PI-positive dead cells was low and there was no difference between CD10-A375 and mock-A375. Under the treatment with the anticancer drugs, however, the number of apoptotic cells increased remarkably. CD10-A375 cells (red line) displayed a lower proportion of Annexin V-positive or Annexin V/PI-positive dead cells induced by anticancer drugs than mock-A375 (blue line), showing stronger resistance. Isotype control is shown with a gray line.