Studies on a growth‐inhibitory peptide derived from alpha‐fetoprotein and some analogs

Abstract

A 34-amino acid synthetic peptide was derived from the third domain of human alpha-fetoprotein, and the peptide was shown to inhibit estrogen-stimulated growth. Under certain conditions, however, the peptide lost growth-inhibitory activity. A biophysical study of the peptide was undertaken with a goal of obtaining completely reliable preparations. The peptide was studied using gel-filtration column chromatography as a function of peptide concentration and age of solution, and was found to exhibit complex aggregation behaviors. During the early period (0-3 h) after dissolving lyophilized peptide into pH 7.4 buffer, solutions were composed mostly of trimers. At higher peptide concentrations (> or = 3.0 g/L), the trimers aggregated extensively to a large aggregate (minimum size approximately 102 peptides). At 5.0-8.0 g/L, these large aggregates increased in size (up to approximately 146 peptides) until trimers were largely exhausted from solution. During the later times (>3 h) after sample preparation, the trimeric oligomer of the peptide dissociated slowly to form dimers for samples at 0.10-3.0 g/L. After their build-up, a very small number of dimers associated to form hexamers. Disulfide bonds stabilized the dimers as indicated by the conversion of dimers to trimers upon the addition of a reducing agent, and the failure of dimers to form in the presence of reducing agent. Reducing agent did not affect trimer or large aggregate formation. Trimers were found to be active in an assay monitoring inhibition of estrogen-stimulated growth, whereas dimers and large aggregates were inactive. The two cysteines in the peptide were modified to either S-methylcysteine or S-(2-aminoethyl)cysteine, and both derivatives showed significant growth-inhibition activity. A serine analog in which both cysteines were replaced had very different aggregation behavior than the cysteine peptide and lacked its growth inhibitory ability. Peptide aggregation is critically important in establishing the ability of the peptide to inhibit growth and have anticancer activity, but the state of its two cysteines is of little influence.

Update of Alpha Fetoprotein Growth-Inhibitory Peptides as Biotherapeutic Agents for Tumor Growth and Metastasis

“…However in GI-101 tumors, P187-treated cells re- The oligomeric forms of GIP in the cytostatic assays also showed differences in their dose effects ( table 2 ). The cyclic form of GIP is a disulfi de-bridged cyclic monomer in solution, whereas GIP in the linear confi guration ( fi g. 1 a, b) is known to exist as a trimer [13][14][15] . Using three Fig.…”

“…In comparison, the same three cancer cell lines showed linear peptide growth inhibition at 10 -4 and 10 -5 M , which rapidly declined thereafter at lower concentrations [12] . The linear peptide is known to be in the trimer/hexamer confi guration [13] . It was also shown that the cyclic versus the linear version of P149 produced different dose responses when applied to the E2-dependent MCF-7 cell foci assay showing an inhibitory effect with low concentrations of linear but not cyclic peptide [9] .…”

“…All peptides were synthesized by F-MOC chemistry using an Applied Biosystems 431A peptide synthesizer (Foster City, Calif., USA) as previously described [13][14][15][16] and purifi ed on reverse-phase high-pressure liquid chromatography. Subsequent binding studies showed that GIP could complex with divalent metal ions showing promise as an antitumor metal chelating agent [16,17] .…”

Effect of α-Fetoprotein and Derived Peptides on Insulin- and Estrogen-Induced Fetotoxicity

“…This concealed site is believed to be exposed by partial unfolding of the native full-length molecule following stress/shock to the fetus. The 34-amino-acid sequence of the concealed site is known, and its synthetic peptide form has been purified, characterized, and assayed for biological activity [7,8]. One major function of the 34-mer peptide has been ascertained to be suppression of growth in neonatal and tumor cells, a function which prompted the name growth-inhibitory peptide (GIP).…”

Development of an Active Site Peptide Analog of α-Fetoprotein That Prevents Breast Cancer

“…Continued efforts to parse the active fragment employed peptide synthesis, beginning with the synthesis of large peptide fragments (51). Extensive studies of a 34-mer peptide that contained the biologic activity (52,53) showed that the large peptide aggregated over time, forming trimers and other disadvantageous aggregates which compromised anti-estrogenic activity. Therefore, it was necessary to identify yet smaller, stable peptides, and Mesfin identified an 8-amino acid fragment of AFP as the smallest peptide that retained full activity (54).…”