Targeting the prostate for destruction through a vascular address

Abstract

Organ specific drug targeting was explored in mice as a possible alternative to surgery to treat prostate diseases. Peptides that specifically recognize the vasculature in the prostate were identified from phage-displayed peptide libraries by selecting for phage capable of homing into the prostate after an i.v. injection. One of the phage selected in this manner homed to the prostate 10-15 times more than to other organs. Unselected phage did not show this preference. The phage bound also to vasculature in the human prostate. The peptide displayed by the prostate-homing phage, SMSIARL (single letter code), was synthesized and shown to inhibit the homing of the phage when co-injected into mice with the phage. Systemic treatment of mice with a chimeric peptide consisting of the SMSIARL homing peptide, linked to a proapoptotic peptide that disrupts mitochondrial membranes, caused tissue destruction in the prostate, but not in other organs. The chimeric peptide delayed the development of the cancers in prostate cancer-prone transgenic mice (TRAMP mice). These results suggest that it may be possible to develop an alternative to surgical prostate resection and that such a treatment may also reduce future cancer risk.

Figure 1

Specific homing of phage to the prostate. (a) Phage selected for prostate homing accumulates specifically in the prostate and the homing is inhibited by soluble peptide. The SMSIARL fUSE5 phage, identified by in vivo screening, was tested for prostate homing. This phage and an irrelevant control phage were injected intravenously to male mice [10⁹ colony forming units (cfu) per mouse] and the phage were rescued from various tissues based on their ability to infect a host bacteria. As indicated, 200 μg of the SMSIARL peptide or a control peptide (CARAC) was included in the injection to test inhibition of SMSIARL phage homing. (b) The SMSIARL peptide directs specific homing of T7 phage to the prostate. The SMSIARL sequence was cloned to the coat protein of the T7. A 1:10 mixture of SMSIARL and nonrecombinant control T7 phage [10¹⁰ plaque-forming units (pfu)] was injected and allowed to circulate for 7 min. Phage was extracted from prostate and brain with buffer (PBS), or a detergent solution (0.5% Nonidet P-40 in PBS) and plated, and 32 colonies were randomly chosen for PCR. The PCR products of SMSIARL and control phage DNA were distinguished on the basis of a size difference in a 4% agarose gel. (Control tissue was brain.)

Figure 2

Immunohistochemical staining of phage within prostate endothelial cells after i.v. injection into mice. SMSIARL-phage preparation was injected intravenously into mice. After 7 min circulation, animals were perfused with PBS, the prostate (a–c), brain (d–f), and various control organs were removed, processed for frozen sectioning, and stained with a polyclonal antibody against T7 phage (FITC; a and d) and CD31 (rhodamine; b and e). Merge with nuclear counterstain with DAPI (c and f). Control organs (kidney, spleen, lung; not shown) were negative for the phage, except for liver and spleen, where the reticuloendothelial tissue traps phage nonspecifically (4). (Magnification: ×400.)

Figure 3

SMSIARL phage binds to endothelium in human prostate. A human prostate tissue section containing both normal and cancerous tissue was overlaid with the SMSIARL phage (10⁹ cfu/ml) and the binding of the phage was detected with anti-M13 phage antibody and peroxidase staining. (a) An overview (×200); (b) a detail from a at a higher magnification (×400). Staining of the endothelium is seen. (c) Overlay with phage that contains no peptide insert produces no endothelial staining. (d) The SMSIARL-phage staining is inhibited when soluble SMSIARL peptide is included in the overlay at 0.3 mg/ml.

Figure 4

Targeted proapoptotic peptide to mouse prostate vasculature causes tissue damage in prostate but not in other tissues. Mice received an i.v. 250 μg injection of the SMSIARL-GG- _D(KLAKLAK)₂ or an equivalent dose of SMSIARL and _D(KLAKLAK)₂ as uncoupled peptides (control-treated mice). The mice were killed 24 h after the injection. Prostates were fixed in paraformaldehyde or glutaraldehyde solution and processed for light microscopy by staining with hematoxylin/eosin (H&E) or electron microscopy. Light microscopy showed focal loss of cell borders and epithelial shedding in the ventral lobe of prostates from the SMSIARL-GG- _D(KLAKLAK)₂ group. (a) H&E-stained micrograph shows massive glandular destruction with nearly complete shedding of the glandular epithelial cells into the lumen. (b) A representative micrograph of normal prostate tissue from a mouse treated with the uncoupled peptide mixture. (Magnification in a and b, ×400.) (c) An electron microscopic image of a single epithelial cell from a SMSIARL-GG-_D(KLAKLAK)₂-group prostate. The cell has sloughed off into the glandular lumen and massive destruction of its organelles is seen. (d) A representative micrograph of normal prostate shows intact cellular structure. (Magnification in c and d, ×6,000.) Light microscopy shows no damage to bladder (e; ×200), heart (f; ×400), kidney (g; ×400), or liver (h; ×400).

Figure 5

Survival of TRAMP mice treated with SMSIARL-GG-_D(KLAKLAK)₂ or control materials. The treatment was initiated at 12 weeks of age. Male mice (ten per group) received i.v. injections of SMSIARL-GG-_D(KLAKLAK)₂ peptide (200 μg per dose), or an equivalent dose of SMSIARL and (KLAKLAK)₂ as uncoupled peptides (control-treated group). The injections were given once a week for a total of ten doses. The mice in the SMSIARL-GG- _D(KLAKLAK)₂ group survived significantly longer than the control mice treated with the uncoupled peptide mixture or with buffer.