Expression and roles of Slit/Robo in human ovarian cancer

Abstract

The Slit glycoproteins and their Roundabout (Robo) receptors regulate migration and growth of many types of cells including human cancer cells. However, little is known about the expression and roles of Slit/Robo in human ovarian cancer. Herein, we examined the expression of Slit/Robo in human normal and malignant ovarian tissues and its potential participation in regulating migration and proliferation of human ovarian cancer cells using two ovarian cancer cell lines, OVCAR-3 and SKOV-3. We demonstrated that Slit2/3 and Robo1 were immunolocalized primarily in stromal cells in human normal ovaries and in cancer cells in many histotypes of ovarian cancer tissues. Protein expression of Slit2/3 and Robo1/4 was also identified in OVCAR-3 and SKOV-3 cells. However, recombinant human Slit2 did not significantly affect SKOV-3 cell migration, and OVCAR-3 and SKOV-3 cell proliferation. Slit2 also did not induce ERK1/2 and AKT1 phosphorylation in OVCAR-3 and SKOV-3 cells. The current findings indicate that three major members (Slit2/3 and Robo1) of Slit/Robo family are widely expressed in the human normal and malignant ovarian tissues and in OVCAR-3 and SKOV-3 cells. However, Slit/Robo signaling may not play an important role in regulating human ovarian cancer cell proliferation and migration.

Fig. 1

Immunohistochemical analysis of Slit2/3 and Robo1/4 in human ovarian cancer tissue microarray. Immunolocalization of Slit2 (A), Slit3 (B), Robo1 (C), and Robo4 (D) was performed as described in “Materials and Methods”. Reddish color indicates positive Slit and Robo staining. For each target protein, representative images from NORM (a), AGCT (b), DISG (c), ADEN (d), TMC (e), YST (f), Mu-ADEN (g), L-Se-ADEN (h), and H-Se-ADEN (i) are shown. For the goat (A, B) or rabbit (C, D) preimmune IgG control, representative images from the L-Se-ADEN are shown. Bar 200 µm. For the semiquantitative analysis of the Slit/Robo staining intensity (E), data are expressed as means ± SEM of the integrated OD. *Differs from NORM (p ≤ 0.05)

Fig. 2

Western blot analysis for Slit2/3 and Robo1/4 in OVCAR-3 and SKOV-3 cells. Cells at 80–90% confluence were harvested and subjected to Western blotting for Slit2/3 and Robo1/4. Different lanes in each individual ovarian cancer cell line represent different passages of cells. Positive controls for Slit2, Slit3, and Robo1/4 are human hepatoblastoma cell lysate, mouse thyroid extract, and HUVE cells, respectively

Fig. 3

Effects of Slit2 on phosphorylation of ERK1/2 and AKT1 in OVCAR-3 and SKOV-3 cells. After serum starvation for 16 h, cells at 80–90% confluence were treated with Slit2 (100 ng/ml) up to 3 h. Proteins were subjected to Western blotting for total (t) or phospho (p) ERK1/2 and AKT1. A representative Western blot from four independent experiments was shown for each target protein. m minutes, h hours

Fig. 4

Effects of Slit2 on SKOV-3 and HUVE cell migration. After serum starvation, cells were seeded in the inserts. Slit2 (100 ng/ml) in 0.5% FBS (for SKOV-3 cells) or in 1% FBS (for HUVE cells) were added into the bottom wells. After 16 h of treatment, the numbers of migrated cells were counted. Cell migration is expressed as means ± SEM fold of the control from four independent experiments. The numbers of migrated cells in the control after 16 h of treatment were 990 ± 66.5 and 376 ± 98.5 for SKOV-3 and HUVE cells, respectively. *Differs from the day 0 control (p ≤ 0.01). Bars 200 µm

Fig. 5

Effects of Slit2 on OVCAR-3 and SKOV-3 cell proliferation. After serum starvation, cells were treated without or with different doses of Slit2 for 4 days. The media were replaced with fresh Slit2 every 48 h. Cell numbers are expressed as means ± SEM from three independent experiments

Fig. 6

Effects of Slit2 on the scratch wound healing in OVCAR-3 and SKOV-3 cells. After serum starvation, confluent cells grown on the 12-well plates were scratched followed by treating cells with Slit2 (100 ng/ml) up to 24 h. The scratch areas (mm²) were determined at time 0, 16, and 24 h. Data are expressed as means ± SEM scratch areas per image from three independent experiments. ND not detectable, Bars 200 µm