Increased potency of the PHSCN dendrimer as an inhibitor of human prostate cancer cell invasion, extravasation, and lung colony formation

Abstract

Activated alpha5beta1 integrin occurs specifically on tumor cells and on endothelial cells of tumor-associated vasculature, and plays a key role in invasion and metastasis. The PHSCN peptide (Ac-PHSCN-NH(2)) preferentially binds activated alpha5beta1, to block invasion in vitro, and inhibit growth, metastasis and tumor recurrence in preclinical models of prostate cancer. In Phase I clinical trial, systemic Ac-PHSCN-NH(2) monotherapy was well tolerated, and metastatic disease progression was prevented for 4-14 months in one-third of treated patients. We have developed a significantly more potent derivative, the PHSCN-polylysine dendrimer (Ac-PHSCNGGK-MAP). Using in vitro invasion assays with naturally serum-free basement membranes, we observed that the PHSCN dendrimer was 130- to 1900-fold more potent than the PHSCN peptide at blocking alpha5beta1-mediated invasion by DU 145 and PC-3 human prostate cancer cells, whether invasion was induced by serum, or by the Ac-PHSRN-NH(2) peptide, under serum-free conditions. The PHSCN dendrimer was also approximately 800 times more effective than PHSCN peptide at preventing DU 145 and PC-3 extravasation in the lungs of athymic mice. Chou-Talalay analysis suggested that inhibition of both invasion in vitro and extravasation in vivo by the PHSCN dendrimer are highly synergistic. We found that many extravasated DU 145 and PC-3 cells go onto develop into metastatic colonies, and that a single pretreatment with the PHSCN dendrimer was 100-fold more affective than the PHSCN peptide at reducing lung colony formation. Since many patients newly diagnosed with prostate cancer already have locally advanced or metastatic disease, the availability of a well-tolerated, nontoxic systemic therapy, like the PHSCN dendrimer, which prevents metastatic progression by inhibiting invasion, could be very beneficial.

Fig. 1

Inhibition of DU 145 and PC 3 cell extravasation into the lungs of nude mice by pretreatment with MCA1187 blocking anti α5β1 MAb. X axes, treatments; Y axes, mean number of extravasated cells per lung (+/− SD). Panel (a). Inhibition of DU 145 extravasation. Panel (b). Inhibition of PC-3 extravasation.

Fig. 2

Hill-Slope plots of the increased invasion-inhibitory potency of Ac-PHSCNGGK-MAP as compared with the Ac-PHSCN-NH₂ peptide with naturally serum-free, selectively permeable basement membranes from sea urchin embryos as invasion substrates. Panel (a). Inhibition of serum–induced DU 145 invasion. Panel (b). Inhibition of serum–induced PC-3 invasion. Panel (c). Inhibition of Ac-PHSRN-NH₂–induced, SF DU 145 invasion. Panel (d). Inhibition of Ac-PHSRN-NH₂–induced, SF PC-3 invasion. X axes, log peptide concentration in nM; Y axes, mean percentages of invaded cells, relative to uninhibited, FBS– or PHSRN–stimulated controls (+/− SD). Black circles, Ac-PHSCNGGK-MAP; black triangles, Ac-PHSCN-NH₂ peptide; black diamonds, Ac-HSPNCGGK-MAP; white circles, Ac-HSPNC-NH₂ peptide; white squares, Ac-PHSCNGGK-Ova.

Fig. 3

Increased potency of Ac-PHSCNGGK-MAP as an inhibitor of α5β1–mediated MMP-1 secretion in vitro by ELISA detection. Panel (a). Inhibition of MMP-1 secretion by DU 145 cells. Panel (b). Inhibition of MMP-1 secretion by PC-3 cells. X axes, treatment groups in triplicate of Ac-PHSCN-NH₂, Ac-PHSCNGGK-MAP, and Ac-HSPNCGGK-MAP at 250 µg/1,000,000 cells; black bars, stimulation with 10% FBS; white bars, stimulation with 250 µg/1,000,000 cells Ac-PHSRN-NH₂. Y axes, mean % MMP-1 activity relative to stimulated control cells (+/− SD).

Fig. 4

Increased extravasation inhibition by Ac-PHSCNGGK-MAP prebinding, relative to the PHSCN peptide. Black circles, Ac-PHSCNGGK-MAP; black squares, Ac-PHSCN-NH₂ peptide. Panel (a). Median-affect plot for DU 145 cells. Panel (b). Median-affect plot for PC 3 cells. Panel (c). PC-3 cell extravasation, typical example of a scored section. Arrows indicate all extravasated cells visible in section.

Fig. 5

Reduced extravascular prostate cancer cells after PHSCNGGK-MAP treatment. Panel (a). Quantitation of intravascular and extravascular DU 145 and PC-3 cells in sectioned mouse lungs. X axis, cell lines; Y axis, mean percentages of cells. Blue bars, intravascular cells; gray bars, extravascular cells. Panels (b and c). Examples of DU 145 cells (b) and PC-3 cells (c) lung sections from untreated (Control) and Ac-PHSCNGGK-MAP treated mice. Arrows, extravascular cells. Stars, intravascular cells.

Fig. 6

Increased inhibition of lung colony formation by Ac-PHSCNGGK-MAP prebinding, relative to the PHSCN peptide. Panel (a). DU 145 colonies. Panel (b). PC 3 colonies. X axis, log (nM), Black bars, Ac-PHSCNGGK-MAP); white bars, Ac-PHSCN-NH₂; dark gray bars, Ac-HSPNCGGK-MAP. Y axis, % Colonies/section as compared to untreated tissue. Panels (c and d). Examples of sectioned lung tissue after DU 145 cell injection (c) or PC-3 cell injection (d) from untreated (Control), 10 ng/ml PHSCNGGK-MAP treated, and 100 ng/ml HSPNCGGK-MAP treated mice.

Fig. 7

Effects of PHSCN dendrimer on clonogenic survival in vitro by DU 145 and PC-3 cells. Panel (a) Clonogenic survival of DU 145 cells. Panel (b) Clonogenic survival of PC-3 cells. X axis, µg per 2 ml well; Y axis, mean number of colonies per well (+/− SD). Black bars, Ac-PHSCNGGK-MAP; dark gray bars, Ac-HSPNCGGK-MAP, light gray bars, Ac-PHSCN-NH₂; white bar, untreated.