The Ac-PHSCN-NH2 Peptide as a Potent Anti-Tumorigenic and Anti-Metastatic Agent for Prostate Cancer
August 1, 2000
The Ac-PHSCN-NH2 Peptide as a Potent Anti-Tumorigenic and Anti-Metastatic Agent for Prostate Cancer
By Donna L. Livant, PhD, and Ameet Upadhyaya
Claiming thousands of lives per year, prostate cancer has emerged as the second most common malignancy among men in the United States, yet there are only limited options for therapy.1 If the cancer is localized, a radical prostatectomy can be curative. Unfortunately, at the time of diagnosis, approximately 60% of prostate cancers are not organ confined.2 For non-organ confined or metastatic prostate cancer, the next option is androgen ablation therapy. Since tumors of the prostate initially require androgen stimulation for growth, such therapy can induce a temporary remission. However, a relapse of the disease is expected after a median of 18 months because of the selection of an androgen-independent clone of cancer cells.3,4 Since androgen withdrawal remains the only therapy that can slow the progression of metastatic prostate cancer, further therapeutic options must be developed to successfully counter the disease at this level of progression.
Background
Effective animal models are necessary to develop therapies for prostate carcinoma. Copenhagen rats engrafted with metastatic, MATLyLu (MLL) cells are one example of a well-tested animal model of androgen-resistant prostate cancer that has been successfully employed.5,6 MLL is a highly invasive, androgen-independent rat prostate carcinoma that, upon injection of a million cells into a rat, culminates in animal death within approximately 25 days due to an overwhelming primary tumor burden;7 or if the primary tumor is excised, leads to animal death due to the presence of lung and lymph node metastases after approximately 40 days.8
Using this animal model, the research focus of this laboratory has centered on the anti-tumorigenic and anti-metastatic potential of the blocked PHSCN (Ac-PHSCN-NH2) peptide. Numerous results demonstrating this peptide’s ability to perform as a therapeutic agent against prostate cancer have been obtained.
From Plasma Fibronectin to the Ac-PHSCN-NH2 Peptide
Plasma fibronectin is a soluble extracellular matrix protein found in plasma, lymph, and interstitial fluid at a concentration range of 0.3-0.5 mg/mL.9 After blood clotting, it is found in serum at reduced concentrations.10 Sea urchin embryos were chosen as the source of basement membranes for in vitro invasion substrates because they contain no endogenous blood or blood proteins and because of their ease of preparation. These basement membranes have been shown to be structurally similar to mammalian basement membranes, and to be selectively permeable to pigment cells during embryogenesis.11,12 Using sea urchin embryo basement membranes as invasion substrates, it was shown that serum was required for in vitro invasion by human and rat metastatic prostate cancer cell lines.13 Further experiments demonstrated that plasma fibronectin was necessary and sufficient to induce invasion because the DU-145 human prostate cancer cell line, which was cultured from a brain metastasis, failed to invade in fibronectin-depleted serum but did invade when fibronectin was replaced.14 Also, addition of fibronectin to serum-free medium stimulated invasion as efficiently as whole serum.15
Subsequent experiments showed that a peptide consisting of the PHSRN synergy sequence, which is located on module 9 of plasma fibronectin’s cell-binding domain and interacts with a specific pocket of the a5 chain of the a5b1 integrin fibronectin receptor, was sufficient to induce DU-145 invasion at concentrations of 1 micromolar or less.15-17 It was rationalized that if DU-145 cells require serum to invade, and if the PHSRN sequence of fibronectin induces DU-145 invasion, then a competitive inhibitor of PHSRN should block both PHSRN-induced and serum-induced invasion. Substitution of arginine with cysteine was shown to form an efficient, competitive invasion inhibitor, the PHSCN peptide. Blocking the termini of the peptide by acetylation and amidation formed a 30-fold more potent invasion inhibitor, the Ac-PHSCN-NH2 peptide.15 The observed increase in activity may have resulted because removing charges at the NH2 and COOH termini allowed the peptide to assume a more active conformation, or because its resistance to exoproteinases was increased.
Ac-PHSCN-NH2 as an Anti-Tumorigenic Agent
Using 19 Copenhagen rats subcutaneously injected with 100,000 MLL cells in a right hind leg, Ac-PHSCN-NH2 was tested in vivo to assess its anti-tumorigenicity. Ten of the rats received intravenous injections of 1 mg Ac-PHSCN-NH2 beginning on the day after MLL cell injection, while the other nine remained untreated. The treatment was administered three times weekly via intravenous tail injections. After 16 days of tumor growth and a total of five injections, the mean volume of the MLL tumors in the treated rats was about 2000-fold less than the mean volume of the MLL tumors in the untreated rats, suggesting that Ac-PHSCN-NH2 may have substantial anti-tumorigenic activity.15
There are two possible explanations for the anti-tumorigenic effects of Ac-PHSCN-NH2. First, it may inhibit MLL tumor growth by stimulating apoptosis. To test for this phenomenon, MLL cells were cultured for 72 hours (the time required for at least 2 cell cycles) in various concentrations of Ac-PHSCN-NH2. After labeling their DNA with propidium iodide, the fraction of sub-G1 (apoptotic) cells was determined by flow cytometry.18 No sub-G1 cells were observed even at concentrations of Ac-PHSCN-NH2 significantly higher than what the tumor may have encountered in vivo.15
The other possible explanation is that Ac-PHSCN-NH2 reduced tumor growth by inhibiting tumor invasion by endothelial cells, thereby limiting tumor vascularization and hence, the supply of nutrients and oxygen to the growing tumors. To explore this effect, the Ac-PHSCN-NH2-treated rat with the largest primary MLL tumor was euthanized. Cryosections of the primary MLL tumor from the Ac-PHSCN-NH2-treated rat and from a typical 2 cm tumor of an untreated rat were immunostained using an anti-rat platelet endothelial cell adhesion molecule (anti-PECAM) monoclonal antibody.19,20 Computing the areas occupied by vasculature in 20 randomly chosen fields from each of the four sections of MLL tumors obtained from the treated and the untreated rats by an image analysis program showed that blood vessel density was reduced 12-fold in the tumor from Ac-PHSCN-NH2-treated rat compared to the tumor from the untreated rat. Since the largest tumor born by an Ac-PHSCN-NH2-treated rat was analyzed, it is likely that at least comparable reduction in the vascularization of the MLL tumors in the other Ac-PHSCN-NH2-treated rats occurred. These results demonstrated that the anti-tumorigenic effects of Ac-PHSCN-NH2 were likely due to the peptide’s anti-angiogenic activity.
Ac-PHSCN-NH2 as an Anti-Metastatic Agent
The experiment exploring the anti-tumorigenic potential of Ac-PHSCN-NH2 was continued in nine treated and in nine untreated rats to measure its effectiveness in inhibiting metastasis. Primary tumors were removed only when they reached a diameter of 2 cm. In the untreated rats, this occurred after 17 days of growth. In the Ac-PHSCN-NH2-treated rats, this did not occur until 30 or 31 days of growth. Thirty-one days after the initial injection of the MLL cells, all rats were sacrificed. Subsequently, the numbers of metastatic MLL colonies found on the surfaces of the fixed lungs of both Ac-PHSCN-NH2-treated and -untreated rats were scored under 10-fold magnification. Additionally, the entire lower right lobe from the fixed lungs of each animal was removed to perform a histological analysis of micrometastasis.
In the Ac-PHSCN-NH2-treated group, four animals had a combined total of nine surface metastatic colonies. The other five treated animals had no visible colonies, for a mean number of 1 surface metastatic colony per treated rat. In contrast, a total of 357 metastatic colonies were observed in the nine animals of the untreated group, for a mean of 39.7 colonies per individual.15 In addition, to demonstrate the sequence specificity of the therapeutic effect, four animals were treated on the same dosage schedule with 1 mg doses of a scrambled peptide, Ac-HSPNC-NH2. In this treatment group, the growth of the primary MLL tumors was as rapid as that observed in the untreated controls. Also, in this group, a total of 927 surface lung metastases were observed for a mean of 231.8 colonies per individual. Thus, systemic Ac-PHSCN-NH2 therapy appeared to be effective at inhibiting tumorigenesis and at preventing metastasis. Furthermore, its effects were shown to be sequence-specific.
As expected, the micrometastasis frequency was also significantly reduced in the Ac-PHSCN-NH2-treated animals with respect to the Ac-HSPNC-NH2-treated and the untreated animals. The entire lower right lobes of six Ac-PHSCN-NH2-treated, six untreated, and four Ac-HSPNC-NH2-treated rat lungs were sectioned and stained with hematoxylin and eosin. Four sections, each separated by 150 microns, were scored for micrometastases in each animal. A total of three micrometastases were found in the six Ac-PHSCN-NH2-treated animals, with a mean number of 0.5 micrometases per individual in the treated group. In contrast, a total of 789 micrometastases in six untreated rats were detected, with a mean number of 131.5 micrometastases per untreated individual. A total of 428 micrometastases were found in the four rats treated with Ac-HSPNC-NH2, with a mean number of 107 micrometastases per individual.15
Evaluation of Anti-Metastatic Activity
To be an effective anti-metastatic agent, Ac-PHSCN-NH2 should be able to inhibit metastasis formation, even in rats that have grown large primary MLL tumors without treatment. To evaluate the anti-metastatic activity of Ac-PHSCN-NH2 in rats with large, untreated MLL tumors, 20 rats were allowed to grow MLL tumors until they reached a mean diameter of 1.9 cm without any treatment. Twenty-four hours after surgical removal of the primary tumors from all rats, 10 randomly chosen rats began to receive thrice-weekly, intravenous Ac-PHSCN-NH2 injections of 1 mg, while the other 10 animals remained untreated. All rats were euthanized two weeks after surgical removal of their primary tumors, and their lungs were scored for both surface metastases and micrometastases, as previously described.
In the treated group, which received a total of six Ac-PHSCN-NH2 injections, surface metastasis was reduced by 99% and micrometastasis by 95%, although treatment did not commence until 24 hours after removal of the primary tumor.15 These results indicate that Ac-PHSCN-NH2 has a substantial direct anti-metastatic effect independent of its anti-tumorigenic activity, and that it may be a particularly effective anti-metastatic agent in cancers that, like prostate cancer, are slow to metastasize.
From a theoretical standpoint, the anti-metastatic effect of Ac-PHSCN-NH2 is an expected consequence of its activity in vitro. Normal epithelial cells like those of the prostate, as well as fibroblasts and endothelial cells, express two key integrins involved in cellular migration that bind plasma fibronectin: a5b1 and a4b1. The interaction of a4b1 with intact fibronectin has been shown to inhibit both invasion and matrix metalloproteinase expression, which otherwise result from the interaction of a5b1 with the PHSRN sequence of fibronectin.21,22 Moreover, the a4b1 receptor has been shown to be absent from the cell membranes of prostate cancer cells.23,24 Thus, in the presence of the intact plasma fibronectin of body fluids, the loss of surface a4b1 receptor may be a proximal cause of the unregulated, constitutively invasive response exhibited by prostate carcinomas. Furthermore, since PHSCN has been shown to inhibit PHSRN-, fibronectin-, and serum-induced invasion in vitro, the anti-angiogenic and anti-metastatic effects of Ac-PHSCN-NH2 are fully consistent with its in vitro activity.
Summary
Ac-PHSCN-NH2 is not the first agent to inhibit tumorigenesis or metastasis of androgen-resistant prostate cancer in animal models. For example, when used in combination, paclitaxel and quinacrine have been shown to reduce DU-145 prostate cancer tumor volume in nude mice by approximately 2.5-fold relative to untreated tumors after two weeks of tumor growth.25 In another anti-tumorigenic therapy, a growth hormone-releasing hormone antagonist also demonstrated some effectiveness in slowing AT-1 prostate tumor growth in rats.26 Also, oral administration of modified citrus pectin has been shown to result in a 40- to 50-fold reduction in the number of MLL metastases in treated vs. untreated rats, without reducing the growth of the primary tumor.5
Thus, Ac-PHSCN-NH2 appears to be uniquely effective in that it dramatically reduces both tumorigenesis and metastasis without demonstrable toxic side effects. Furthermore, its anti-metastatic effect appears to be independent of its anti-tumorigenic activity.
Since Ac-PHSCN-NH2 is a competitive inhibitor of the a5b1 integrin, it may be effective against cancers arising in tissues other than the prostate. Any cancer in which surface expression of the a5b1 receptor occurs in the presence of reduced levels of a4b1 may be a potential candidate for therapy with this agent. For example, preclinical results from our laboratory indicate that systemic Ac-PHSCN-NH2 therapy is equally effective at inhibiting tumorigenesis and metastasis in nude mice injected with metastatic human breast, as well as human prostate cancer cells.
Since plasma fibronectin fragmentation occurs as a result of blood clotting, and fibronectin fragments bearing the PHSRN sequence are known to diffuse from wounds and to stimulate invasion by normal epithelial, mesenchymal, and blood cells involved in wound healing, it is intriguing to consider that the constitutively invasive phenotype of many types of cancer cells may arise from the aberrant expression or response of integrin fibronectin receptors to the intact plasma fibronectin of all body fluids.27-29 Ultimately, this type of research may lead to an effective paradigm with which to devise potent, non-toxic therapeutic agents for combating a variety of commonly occurring cancers. (Ameet Upadhyaya is a Research Associate in the laboratory of Dr. Livant in the Department of Cell & Developmental Biology, University of Michigan Medical School, Ann Arbor, MI.)
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