An 8-mer peptide (EMTOVNOG) derived from ␣-fetoprotein was compared with tamoxifen for activity against growth of human breast cancer xenografts implanted in immune-deficient mice. Both peptide and tamoxifen prevented growth of estrogen-receptorpositive MCF-7 and T47D human breast cancer xenografts. A subline of MCF-7, made resistant to tamoxifen by a 6-month exposure to this drug in culture, was found to be resistant to tamoxifen in vivo. Peptide completely prevented the xenograft growth of this tamoxifen-resistant subline of MCF-7. Neither peptide nor tamoxifen was effective in slowing the xenograft growth of the estrogen-receptor-negative MDA-MB-231 human breast cancer. A worrisome side effect of tamoxifen is its hypertrophic effect on the uterus. In this study, tamoxifen was shown to stimulate the growth of the immature mouse uterus in vivo, and the peptide significantly inhibited tamoxifen's uterotrophic effect. The mechanism of action of peptide is different from that of tamoxifen in that the peptide does not interfere with the binding of ) have shown that elevated levels of AFP during pregnancy are associated with a subsequent reduction in lifetime risk for breast cancer, and Jacobson has hypothesized that this should be caused by a diminution in estrogen-dependent breast cancers (6). Sonnenschein et al. (7) have shown in rats that an AFP-secreting hepatoma prevents the growth of an estrogen-dependent breast cancer in the same rat. Finally, we have shown that AFP purified from a human hepatoma culture and then injected into tumor-bearing, immune-deficient mice stopped the growth of estrogenreceptor-positive (ERϩ), but not estrogen-receptor-negative (ERϪ), human breast cancer xenografts in these mice and did so by a mechanism different from that of tamoxifen (1).More recently, we have identified the active site of AFP responsible for its antiestrotrophic activity (8). It consists of amino acids 472-479 (EMTPVNPG), an 8-mer sequence in the 580-aa AFP molecule. We have synthesized this 8-mer peptide, modified it by substituting hydroxyproline (O) for proline (P) for the purpose of stabilization, and have shown that this new analog (EMTOVNOG) is stable during long-term storage and, like AFP, has the ability to inhibit estrogen-stimulated growth of breast cancer cells in culture and estrogen-stimulated growth of the uterus in immature mice (8,9). Having the stable analog in hand, a purpose of the study described herein was to determine whether this peptide has anti-breast cancer activity in vivo like its parent protein, AFP. A second purpose of the study was to compare the activities of this peptide with those of tamoxifen.

The peptide, EMTPVNPG, derived from alpha-fetoprotein, inhibits estrogen-stimulated growth of immature mouse uterus and estrogen-dependent proliferation of human breast cancer cells. However, the biological activities of the peptide diminish over time in storage, even when in the lyophilized state, probably because of peptide aggregation through hydrophobic interaction among monomers. Two analogs of EMTPVNPG were designed with the intent of minimizing aggregation and retaining biological activity during prolonged storage. EMTOVNOG, where O is 4-hydroxyproline, is a linear peptide generated by substituting 4-hydroxyproline for the two prolines, thereby increasing peptide hydrophilicity. This analog exhibited a dose-dependent inhibition of estrogen-stimulated growth of immature mouse uterus similar to that of EMTPVNPG (maximal activity at 1 microg/mouse). A second analog, cyclo-(EMTOVNOGQ), a hydrophilic, cyclic analog with increased conformational constraint, was as potent as the other peptides in its inhibition of estrogen-dependent growth of immature mouse uterus, and had an expanded effective dose range. Both linear and cyclized hydroxyproline-substituted analogs exhibited indefinite shelf-life. Furthermore, both analogs inhibited the estrogen-dependent growth of MCF-7 human breast cancer growing as a xenograft in SCID mice. These analogs may become significant, novel agents for the treatment of breast cancer.

Data from the Cancer and Steroid Hormone Study, a large nationwide population-based case-control study conducted in the United States in 1980-1982, were analyzed to investigate whether pregnancies ending in a multiple birth affect the risk of subsequent breast cancer. The cases were 3,918 parous women who were aged 20-54 years and newly diagnosed with breast cancer; controls were 4,047 parous women selected randomly from the same geographic areas as the cases. Multiple births were reported by 118 cases and 161 controls. After adjustment for other reproductive variables, having a multiple last birth was found to be protective against breast cancer (odds ratio (OR) = 0.60, 95% confidence interval (CI) 0.43-0.85), whereas having a multiple birth prior to the last birth was not (OR = 1.11, 95% CI 0.79-1.57). To the authors' knowledge, this study is the first investigation to report such a protective effect, and thus the finding warrants replication. One mechanism that might account for the effect involves the increased output of alpha-fetoprotein by multiple fetal livers.