Figures & data
Figure 1 SHP2 overexpression in human ovarian cancer cell lines.
Notes: (A) Expression of SHP2 in normal human ovarian cells and ovarian cancer cell lines. Lanes: 1, IOSE80; 2, SKOV3; 3, A2780; 4, Ho-8910PM; 5, OVCA420; 6, OVCA429; and 7, OV-01063 cells. (B) Quantification of the relative intensities of the bands obtained from the Western blot presented in (A). *P<0.05 and **P<0.01 compared with normal human ovarian cells (lane 1).
Figure 2 SHP2 overexpression in human ovarian tumor tissues.
Notes: (A) Representative images of immunohistochemical staining of human ovarian tissues (magnification, 400×). Immunohistochemical staining was performed to evaluate the SHP2 expression levels in 60 human ovarian cancer tissues (3+) and 60 matched normal ovarian tissues (1+). (B) Western blot analysis of SHP2 protein expression in human ovarian tissue specimens. (C) A semi-quantitative Western blot analysis of human ovarian tissue samples revealed that the levels of the SHP2 protein were significantly elevated in tumor tissues compared with normal tissues. Actin was used as an internal control. **P<0.01 compared with normal tissues, *P<0.05.
Abbreviations: N, normal; T, tumor.
Abbreviations: N, normal; T, tumor.
Table 1 Expression of SHP2 in normal ovarian tissues and epithelial ovarian cancer
Table 2 Correlation between SHP2 expression and clinicopathological factors in epithelial ovarian cancer
Figure 3 SHP2 overexpression enhances the proliferation of A2780 cells.
Notes: (A) SHP2-positive foci in 3 wells from each cell line. (B) Anchorage-independent growth was assayed by evaluating colony growth in soft agar. Representative images are shown. The colonies formed by the SHP2-expressing cells are larger in size. Representative images of randomly selected cells from the experimental groups are shown. (C and D) The number of clones in each experimental group was counted. Each experiment was repeated a minimum of 3 times. The data shown here are representative of at least 3 independent experiments with similar results. Significant differences are indicated by asterisks (*P<0.05 and **P<0.01).
Figure 4 SHP2 overexpression enhances the migration, invasion and proliferation of A2780 cells.
Notes: (A) Wound-healing assays were conducted to evaluate cell migration. The width of the scratch indicated the migratory ability of the tumor cells. (B) Crystal violet staining of the membrane used in the Boyden chamber migration assay. (C) The invasive cells were counted using high-power microscopy images at a magnification of 200×. **Invasive capacity of A2780 cells. (D) Proliferative capacity of A2780 cells (*P<0.05 and **P<0.01).
Figure 5 SHP2 overexpression is associated with increased resistance to the chemotherapeutic agent paclitaxel.
Notes: (A) Flow cytometry analysis of apoptosis in A2780 cells incubated with Taxol (3 μg/mL) for 24 h. The number of cells was calculated at the same time point, and the data are expressed as the growth rate of SHP2-overexpressing cells compared with the vehicle control group (**P<0.01). (B) SHP2 and caspase-3 expression. The expression levels were determined using Western blot assays. (C) Quantification of the relative intensities of the bands obtained from the Western blot assay presented in (B). *P<0.05 and **P<0.01 compared with the control group.
Figure 6 SHP2 overexpression promotes tumor growth and tumor angiogenesis in a mouse xenograft model.
Notes: (A) Representative images of the ovarian tumors derived from the SHP2-overexpressing (lower row) and vector control (upper row) groups. The images were captured immediately after the mice were sacrificed. (B) The tumor size was monitored every 3 days after the cell injections. (C) The solid tumors were removed and (D) weighed after the mice were sacrificed. The data are presented as mean ± SEM (n=4 per group) (*P<0.05).
Figure 7 Effect of SHP2 overexpression on tumor metastasis.
Notes: A2780 cells were injected into nude mice. The mice were sacrificed 50 days later and tumor metastasis to distant organs was subsequently assessed. Anatomical images of liver metastasis in mice injected with the SHP2-overexpressing (A) or vector control (B) cells. Lower panel: metastases in the resected livers. (C) Cell morphology was evaluated using H&E staining; a:SHP2 group; b:Vector group. Liver tissues were fixed, sectioned, and stained with H&E, as described in the “Materials and Methods” section. A representative image of metastatic nodes in the livers of mice injected with SHP2-overexpressing cells is shown (magnification, 400×). A minimum of six regions from each tumor were examined.
Table 3 Effect of SHP2 on tumor (A2780) metastases in different organs of mice bearing xenografted tumors
Figure 8 SHP2 overexpression enhances cell migration and invasion by increasing phospho-AKT levels in ovarian cancer cells.
Notes: (A) Cultured cells were serum-starved for 24 h and stimulated with RPMI 1640 medium supplemented with 10% FBS for 2 h. The levels of the E-cadherin, vimentin, and β-actin proteins were evaluated by Western blotting. (B) Densitometry analysis of the bands from the Western blot using the ImageJ program. The levels of the E-cadherin and vimentin proteins were normalized to the levels of the β-actin protein. (C) A2780 cells were serum-starved overnight and subsequently incubated with EGF (5 ng/mL) for 10 or 20 min. The levels of the p-AKT, AKT, and β-actin proteins were evaluated by Western blotting. (D) Densitometry analysis of the bands from the Western blot assays using the ImageJ program. The levels of the p-AKT protein were normalized to the levels of the β-actin protein. The experiment was repeated 3 times.
Abbreviation: FBS, fetal bovine serum.
Abbreviation: FBS, fetal bovine serum.
