Figure 1.
Troglitazone prevents HGF-induced cell surface-directed lysosome trafficking, cathepsin B secretion, and invasion.
A) DU145 prostate tumor cells were treated with 10 µM Troglitazone and/or HGF overnight, followed by I.F. microscopy to visualize lysosomes (red), actin (green), and nuclei (blue). Troglitazone also causes lysosomes to cluster juxtanuclear to the cell nuclei. B) Quantitation of the spatial distribution of lysosomes is shown as mean distance from individual cell nuclei for each treatment condition. Error bars represent the s.e.m of 30 cells from at least three independent experiments. C) DU145 cells were treated for 16 hrs with Troglitazone and/or HGF and the amount of Cathepsin B secreted into the culture media was detected utilizing a cathepsin B activity assay (see methods and materials). D) DU145 cells were seeded onto Matrigel-coated transwell inserts and allowed to invade for 24 hrs. Treatments were added where indicated to both the top and bottom of the insert. *Statistical significance (p<0.001) versus control; **Statistical significance (p<0.01) versus control. Scale bars: 10 µm.
Figure 2.
The GTPase Rab7 is necessary for Troglitazone-mediated prevention of HGF-induced cell surface-directed lysosome trafficking, cathepsin B secretion, and invasion.
A) I.F. microscopy was performed on DU145 prostate tumor cells expressing either a non-target (scrambled) shRNA or a Rab7-directed shRNA to visualize lysosomes (red), actin (green), and nuclei (blue). Rab7 shRNA expression prevents Troglitazone-induced juxtanuclear lysosome clustering and causes lysosomes to traffic to the cell surface independent of HGF. B) Quantitation of the spatial distribution of lysosomes is shown as mean distance from individual cell nuclei for each treatment condition. Error bars represent the s.e.m of 30 cells from at least three independent experiments. C) NT and Rab7 shRNA expressing DU145 cells were treated for 24 hrs with Troglitazone and/or HGF and the amount of Cathepsin B secreted into the culture media was detected utilizing a cathepsin B activity assay (see methods and materials). Cathepsin B secretion is increased and Troglitazone no longer prevents secretion in the Rab7 knockdown cells. Inset indicates LAMP-1 and Rab7 expression in the nontarget (NT) or Rab7 shRNA (KD) stable cell lines by Western blot. D) NT and Rab7 shRNA expressing DU145 cells were seeded onto Matrigel-coated transwell inserts and allowed to invade for 24 hrs. Treatments indicated were added to both the top and bottom of the insert. *Statistical significance (p<0.001) versus NT control; **Statistical significance (p<0.01) versus NT control. Scale bars: 10 µm.
Figure 3.
Rab7 shRNA expressing tumors grow larger, demonstrate increased cell proliferation, and decreased apoptosis in vivo.
A) SCID/Bg mice were injected with 2×106 NT shRNA (n = 6) or Rab7 shRNA (n = 7) expressing DU145 cells s.c. Tumors became measureable by day 41 and were measured via digital calipers twice per week. Mice were sacrificed once the largest tumor reached 1.4 cm2. Data are expressed as cubic millimeter volume. Error bars represent s.e.m. of 6 and 7 tumors respectively. *Statistical significance (p<0.05) versus NT shRNA tumors; ** Statistical significance (p<0.001) versus NT shRNA tumors. B) Immunohistochemistry was performed on formaldehyde fixed tumor tissue (see methods and materials). Cell proliferation was assessed using Ki-67 as a marker and apoptotic cells were visualized using Cleaved Caspase-3 as a marker. C) IHC was quantitated by counting Ki-67 or Cleaved Caspase-3 positive cells. Three random fields per tumor were manually counted. The number of positively staining cells were graphed. *Statistical significance (p<0.01) versus NT shRNA tumors; **Statistical significance (p<0.05) versus NT shRNA tumors. Scale bars: 100 µm.
Figure 4.
Rab7 shRNA expressing tumors demonstrate increased cell invasion into surrounding murine tissue.
H&E stained s.c. tumor cross sections with the surrounding murine tissue and skin shown adjacent to the tumor. Rab7 knockdown tumors display increased infiltration into the tissue layer underlying the skin compared to control tumors. In addition, the tissue displays increased disorder and loss of integrity surrounding the Rab7 knockdown tumors. Scale bar: 100 µm.
Table 1.
Differential regulation of Rab7 in tumor biopsy specimens by microarray analysis.
Figure 5.
Reduced Rab7 expression results in increased c-met expression and enhanced signaling.
A) Nontarget (NT) vector control or Rab7 shRNA expressing DU145 cells were exposed to HGF for the indicated times. Whole cell lysates were collected and c-met, Akt, and Erk phosphorylation was detected by Western blotting. B) Cells were treated with HGF for the indictated time periods and levels of total c-Met protein were determined by Western blot analysis. Densitometry from three independent experiments was used for graphical representation of c-Met loss over time. C) Cells were treated with the indicated concentrations of HGF for 30 minutes followed by Western blot analysis of the indicated proteins. D) Protein lysates of xenograft tumors were harvested and levels of total c-met and Rab7 protein were detected by Western blot. The bar graph represents the average c-met and Rab7 expression of seven tumors per treatment group as determined by densitometry of Western blot. * = p<0.0007 compared to NT shRNA Rab7. ** = p<0.03 compared to NT shRNA c-met. Error bar represent SEM.