It has been demonstrated that invasive prostate cancer cells have a distinct integrin and cell adhesion molecule profile that allows for increased metastatic capabilities [8–10
]. Expression of these integrins is thought to mediate cell adhesion with specific extracellular basement membranes such as laminin, vitronectin, and fibronectin and facilitate cell migration and invasion through specific pathways such as the focal adhesion kinase pathway [11
]. Specific integrin pairs are also implicated in forming cell to cell (tumor cell to tumor cell or tumor cell to endothelial cell) interactions, required for tumor cell metastasis [12,13
]. Integrins that recognize the canonical tripeptide sequence, RGD, found in a variety of extracellular matrix proteins, are thought to potentially mediate prostate cell interactions with endothelial cells [12
]. The DU145 cell has specifically been shown to express the α5
integrin pair, which recognizes the RGD sequence [9
Plasma fibronectin is a soluble extracellular matrix protein found in plasma, lymph, and interstitial fluids [6
]. DU145 and MAT-Ly-Lu cells in serum-free medium can be stimulated to invade through a naturally occurring serum-free basement membrane, SU-ECM, by the addition of plasma fibronectin [4
]. Livant et al. demonstrated that the PHSRN sequence of plasma fibronectin, which maps to the cell-binding domain, was sufficient for inducing invasion of these cells [4
]. Furthermore, they demonstrated that this is due to PHSRN binding to and stimulating the cell through the α5
receptors on the cell surface. In vivo
, prostate cells may also be induced to invade locally by liberation of fibronectin by prostate-specific antigen (PSA) [14
Substitution of the arginine residue in the PHSRN sequence with cysteine forms a competitive inhibitor of basement membrane invasion. The PHSCN peptide can effectively block serum-free, PHSRN-induced invasion of human DU145 and rat MAT-Ly-Lu prostate carcinoma cells in vitro
. Blocking the amino and carboxyl ends of the peptide by acetylation and amidation, respectively, increases its inhibitory activity by 30-fold in serum-containing medium. Taken together, these data led to the hypothesis that Ac-PHSCN-NH2
may be useful in preventing prostate carcinoma metastasis in vivo
, due to its anti-invasive properties. Rats that were systematically administered peptide beginning 24 hours after inoculation with MLL cells had significantly fewer lung metastases compared with rats treated with either saline alone or Ac-HSPNC-NH2
scrambled peptide [4
We, thus, surmised that administration of Ac-PHSCN-NH2 after resecting a primary human prostate xenograft might reduce tumor recurrence and metastases by blocking invasion. Athymic nude mice that had tumors of approximately 900 mm3 began Ac-PHSCN-NH2 24 hours after tumor cell injection. None of the Ac-PHSCN-NH2 had tumor at the site of surgery. All of the untreated animals succumbed to either tumor recurrence and/or metastasis within 17 weeks postsurgery. The peptide-treated animals remained disease free 30 weeks after surgery and had no evidence of disease at either the primary or secondary sites. Microscopic analysis of representative lung sections demonstrated 18-fold greater numbers of micrometastases in the untreated animals compared with the treated animals.
As suggested in previous experiments, the presence of micrometastasis suggest that metastasis could be occurring before surgery. However, metastases could also occur due to, or be enhanced by the “spilling” of tumor cells into the blood or lymph during surgical intervention. Given the observation that tumor recurrences reached a large volume much faster than the primary tumors, it is possible that tumor growth is enhanced by physical injury and proximity to a wound-healing process where brisk angiogenesis is occurring. Likewise, circulating factors could also stimulate tumor cell invasion [15–17
treatment is effective in controlling the growth of recurrences and distant metastases, even if the above processes are in effect.
Similar results were observed when Ac-PHSCN-NH2 was combined with the antiangiogenic compound TM. TM exerts its antiangiogenic effects by sequestering dietary and circulating copper, forming a tripartite complex with copper and protein that is eliminated in the bile and urine. Because copper appears to be required for activation of the angiogenic switch in nascent tumors as well as for sustained angiogenesis in bulky tumors, TM-treated animals produced a significant difference in tumor growth rates relative to the untreated tumors reaching resectable size (900 mm3), with the latter reaching the target size by 12 weeks whereas 1 of 5 of the TM-treated tumors never reached the target resectable size at all.
Most strikingly, the time to tumor resection was very different between the treatment groups and the control. The times to tumor resection increased in the following order: TM+Ac-PHSCN-NH2>TM>Ac-PHSCN-NH2>untreated. Interestingly, in each of the TM-treated groups there was one animal whose tumor never reached a resectable size. Also, the Ac-PHSCN-NH2 tumors appeared to be more defined than the untreated or TM-treated tumors. The untreated tumors in particular had more diffuse contours and were visibly vascularized. TM alone was protective against tumor recurrence, albeit to a slightly lesser extent than Ac-PHSCN-NH2 alone, with 80% of the TM-treated animals being recurrence free compared with 100% of the Ac-PHSCN-NH2-treated animals. Most significantly, all of the treatment groups survived disease free for longer than 30 weeks, compared with the control group that succumbed to disease within 16 weeks postsurgery.
Taken together, these data strongly suggest that Ac-PHSCN-NH2 alone, or in combination with TM, may provide a highly effective, tolerable, long-term treatment for the metastatic and recurrence events of prostate cancer in human xenografts postsurgery. Postsurgical administration of these compounds, each alone or in combination, can effectively prevent prostate cancer recurrence and metastasis. Because the Ac-PHSCN-NH2 is so far available only in injectable form, it is very useful to consider combining or alternating therapy with an oral, nontoxic global inhibitor of angiogenesis such as TM, which alone has significant activity in the prevention of metastasis, has the added advantage of impairing primary tumor growth, and is amenable to long-term administration.