Vitamin D binding protein-macrophage activating factor directly inhibits proliferation, migration, and uPAR expression of prostate cancer cells

Abstract

Background: Vitamin D binding protein-macrophage activating factor (DBP-maf) is a potent inhibitor of tumor growth. Its activity, however, has been attributed to indirect mechanisms such as boosting the immune response by activating macrophages and inhibiting the blood vessel growth necessary for the growth of tumors.

Methods and findings: In this study we show for the first time that DBP-maf exhibits a direct and potent effect on prostate tumor cells in the absence of macrophages. DBP-maf demonstrated inhibitory activity in proliferation studies of both LNCaP and PC3 prostate cancer cell lines as well as metastatic clones of these cells. Flow cytometry studies with annexin V and propidium iodide showed that this inhibitory activity is not due to apoptosis or cell death. DBP-maf also had the ability to inhibit migration of prostate cancer cells in vitro. Finally, DBP-maf was shown to cause a reduction in urokinase plasminogen activator receptor (uPAR) expression in prostate tumor cells. There is evidence that activation of this receptor correlates with tumor metastasis.

Conclusions: These studies show strong inhibitory activity of DBP-maf on prostate tumor cells independent of its macrophage activation.

Figure 1. DBP-maf inhibits tumor cell migration.

LNCaP (A), LNCaPLN3 (B), PC3M (C) or PC3MLN4 (D) cells were added (150,000/well) to the top chamber of a modified Boyden chamber (+/− DBP-maf) with 10% FBS in the bottom chamber. After 6 hours cells were removed that had not migrated and remaining cells were quantitated using an acid phosphatase assay. Results were normalized to control. Experiments were performed a minimum of three times and error is shown as +/− SD. Compared to cell growth without DBP-maf, adding DBP-maf had a statistically significant overall reduction of cell migration at 30% (P = 0.0003) for the combined four tumor cell types. Individual significant reduction rates were found with each of these tumor cell types. Compared to control, significant reduction was seen with DBP-maf at (A) 20% P = 0.0022 (B) 20% P = 0.0029 (C) 10% P = .0045 (D) 30% P = .0094. n = 3.

Figure 2. A DBP-maf peptide inhibits tumor cell migration.

LNCaP (A), LNCaPLN3 (B), PC3M (C) or PC3MLN4 (D) cells were added (150,000/well) to the top chamber of a modified Boyden chamber (+/− DBP-maf) with 10% FBS in the bottom chamber. After 6 hours cells were removed that had not migrated and remaining cells were quantitated using an acid phosphatase assay. Results were normalized to control. Experiments were performed a minimum of three times and error is shown as +/− SD. Compared to migration without DBP-maf, adding DBP-maf had a statistically significant reduction of migration at 40% (P<0.0001) for the combination of all four tumor cell types. Individual significant reduction rates were found with each of these tumor cell types. Compared to control, significant reduction was seen with DBP-maf at (A) 30% P = 0.0038 (B) 40% P = 0.0016 (C) 20% P = .0038 (D) 40% P = .0005. n = 3.

Figure 3. DBP-maf inhibits tumor cell proliferation.

LNCaP (A), LNCaPLN3 (B), PC3M (C) or PC3MLN4 (D) cells were seeded in 24 well dishes overnight, then medium +/− DBP-maf was added with 1% FBS. After 72 hours cells were quantitated using an acid phosphatase assay. Results were normalized to control. Experiments were performed a minimum of three times and error is shown as +/− SD. Compared to control, significant reduction was seen with DBP-maf at (A) 50% P = 0.0001 (B) 50% P = 0.0001 (C) no significant reduction (D) 40% P = .0073. n = 3.

Figure 4. DBP-maf inhibits expression of uPAR in LNCaPLN3 cells.

LNCaP and LNCaPLN3, cells were treated with DBP or DBP-maf (0.001 and 1 µg/mL) and incubated for 24 hours then harvested. RT products (cDNA), identified as uPAR1, 2, and 3, were amplified by real-time quantitative PCR.

Figure 5. DBP-maf inhibits expression of uPAR in PC3M cells.

PC3M, and PC3MLN4 cells were treated with DBP or DBP-maf (0.001 and 1 µg/mL) and incubated for 24 hours then harvested. RT products (cDNA), identified as uPAR1, 2, and 3, were amplified by real-time quantitative PCR. p<0.05.

Figure 6. DBP-maf peptide does not inhibit expression of uPAR in PC3M or LNCaPLN3 cells.

LNCaPLN3 (A) and PC3M cells (B) were treated with DBP or DBP-maf (0.001 and 1 µg/mL) and incubated for 24 hours then harvested. RT products (cDNA), identified as uPAR1, 2, and 3, were amplified by real-time quantitative PCR. p<0.05.

Figure 7. DBP-maf inhibits protein expression of uPAR.

LNCaP, LNCaPLN3, PC3M, and PC3MLN4 were treated with DBP or DBP-maf and incubated for 24 hours (A) then harvested and immunoblotted using an anti-uPAR antibody. LnCaPLN3 cells at 72 hours (B). p<0.05.