Influence on antiproliferative activity of structural modification and conjugation of gonadotropin-releasing hormone (GnRH) analogues.

The effect of various GnRH analogues, and their conjugates on proliferation, clonogenicity and cell cycle phase distribution of MCF-7 and Ishikawa human cancer cell lines was studied. GnRH-III, a sea lamprey GnRH analogue reduced cell proliferation by 35% and clonogenicity by 55%. Structural modifications either decreased, or did not alter biological activity. Conjugation of GnRH analogues including MI-1544, MI-1892, and GnRH-III with poly(N-vinylpyrrolidone-co-maleic acid) (P) through a tetrapeptide spacer GFLG(X) substantially increased the inhibitory effect of the GnRH analogues. The conjugate P-X-GnRH-III induced significant accumulation of cells in the G2/M phase; from 8% to 15.6% at 24 h and 9.8% to 15% at 48 h. It was concluded that conjugation of various GnRH analogues substantially enhanced their antiproliferative activity, strongly reduced cell clonogenicity and retarded cell progression through the cell division cycle at the G2/M phase.

Effects of methylacetylenic putrescine, an ornithine decarboxylase inhibitor and potential novel anticancer agent, on human and mouse cancer cell lines.

Sensitivity of several human and mouse cancer cell lines to methylacetylenic putrescine (MAP) was evaluated using clonogenic, sulforhodamine B and cell counting assays. The effects of MAP on cell morphology, cell cycle phase distribution and changes in polyamine metabolism of xenografted MCF-7 and MDA-MB-231 human mammary tumor cells were also investigated. On the basis of IC50 values, BHT-101 human thyroid carcinoma cells were the most sensitive (9 micrograms/ml), followed by P388 mouse lymphoma (32 micrograms/ml), MCF-7 (48 micrograms/ml) and MDA-MB-231 (110 micrograms/ml) human breast carcinoma cell lines. MAP treatment led to accumulation of P388 cells in G1 phase. At higher doses, the cytoplasm of the cells became vacuolated followed by apoptosis. The foamy cytoplasm may suggest a rare type of cell death (Clarke III type) called non-apoptotic programmed cell death. MAP treatment resulted in a total inhibition of ornithine decarboxylase (ODC) activity with a concomitant decrease of intracellular polyamine (mostly putrescine and spermidine) content in the breast cancer cells, whilst the spermine concentration was shown to increase. MAP proved at least 10 times more potent than the formerly studied DL-alpha-difluoromethylornithine making it an attractive candidate for clinical testing.

Gonadotropin-releasing hormone analogue conjugates with strong selective antitumor activity.

Conjugation of gonadotropin-releasing hormone (GnRH) analogues GnRH-III, MI-1544, and MI-1892 through lysyl side chains and a tetrapeptide spacer, Gly-Phe-Leu-Gly (X) to a copolymer, poly(N-vinylpyrrolidone-co-maleic acid) (P) caused increased antiproliferative activity toward MCF-7 and MDA-MB-231 breast, PC3 and LNCaP prostate, and Ishikawa endometrial cancer cell lines in culture and against tumor development by xenografts of the breast cancer cells in immunodeficient mice. MCF-7 cells treated with P-X-1544 and P-X-1892 displayed characteristic signs of apoptosis, including vacuoles in the cytoplasm, rounding up, apoptotic bodies, bleb formation, and DNA fragmentation. Conjugates, but not free peptides, inhibited cdc25 phosphatase and caused accumulation of Ishikawa and PC3 cells in the G2/M phase of the cell cycle after 24 h at lower doses and in the G1 and G2 phases after 48 h. Since P-X-peptides appear to be internalized, the increased cytotoxicity of the conjugates is attributed to protection of peptides from proteolysis, enhanced interaction of the peptides with the GnRH receptors, and/or internalization of P-X-peptide receptor complexes so that P can exert toxic effects inside, possibly by inhibiting enzymes involved in the cell cycle. The additional specificity of P-X-peptides compared with free peptides for direct antiproliferative effects on the cancer cells but not for interactions in the pituitary indicates the therapeutic potential of the conjugates.

Synthesis of gonadotropin-releasing hormone III analogs. Structure-antitumor activity relationships.

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.

Effect of gonadotropin-releasing hormone analogs and their conjugates on gonadotropin-releasing hormone receptor--positive human cancer cell lines.

The purpose of the present investigation was to develop new gonadotropin-releasing hormone (GnRH) antagonists and to increase their stability and antitumor effect by conjugation with carrier macromolecules. Antitumor effect was evaluated using clonogenic assay, cell counting for antiproliferation, and sulforhodamine B method. The presence of GnRH-binding sites in human cancer cell lines (MCF-7, MDA-MB-231, Ishikawa, LNCaP) was proved. The direct growth inhibition of tumor cell lines is achieved with relatively high analog concentrations (10(-10)- 10(-5) M). We have developed new GnRH analogs of human and chicken origin. MI-1544 (Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10)GnRH and the chicken GnRH antagonist MI-1892 (Ac-D-Trp1,3, D-Cpa2, Lys5, [beta-Asp(DEA)]6, Gln8, D-Ala10)-GnRH have stronger direct antitumor properties than the agonists. The antagonists inhibited proliferation of GnRH receptor-positive human cancer cell lines by 28 to 38%. GnRH peptide analogs were coupled with macromolecules through biodegradable groups, to enhance their antitumor effects. The antagonists reduced survival of MCF-7 and MDA-MB-231 cells by 38 to 48% and 20 to 41%, respectively. They showed less activity against human endometrial and prostate cancer cells (10-20%). The copolymer (P) as polyanionic carrier molecule reached only 15 to 20% survival reduction in all cell lines. However, the copolymer GnRH antagonist conjugates P-X-1892 and P-X-1544 killed 95 to 98% of cells at doses corresponding to the GnRH analog concentration. These compounds having antitumor activity could be tried for the treatment of prostate, breast, and endometrium cancer.

Synthesis of GnRH analogs having direct antitumor and low LH-releasing activity.

New chicken I GnRH agonists and antagonists have been synthesized and tested for their biological activities. The common feature of these analogs was that the molecules had a beta-L-aspartyl residue inserted in position 6. The agonist bound to the pituitary still had low endocrinological activity. On the other hand, it exhibited direct antitumor effect in in vitro assays. The endocrinological activity of the antagonist was low; however, it showed potent, direct antitumor activity. These observations might lead to the development of new GnRH analogs with selective antitumor effect.

Antitumour effect of a gonadotropin-releasing-hormone antagonist (MI-1544) and its conjugate on human breast cancer cells and their xenografts.

Our gonadotropin-releasing hormone (GnRH) antagonist analogue MI-1544 ([Ac-D-Trp1,3,D-Cpa2,D-Lys6,D-Ala10]GnRH) was developed as a potential contraceptive material, because it decreased the luteinizing hormone level without unfavourable side-effects. The antagonist was covalently bound to poly[Lys-(Ac-Glu0.96-DL-Ala3.1)] (AcEAK)-a branched polypeptide having a polylysine backbone--resulting in a MI-1544-AcEAK conjugate. According to our in vitro experiments the MI-1544 induced a 33%-35% decrease in cell numbers of MCF-7 and MDA-MB-231 human breast cancer cell lines at a dose of 30 microM. The biodegradable polymeric carrier, AcEAK, alone inhibited cell proliferation by only 13%-15%, while the MI-1544-AcEAK conjugate, applied at the same dose, was capable of producing 45%-50% inhibition of cell proliferation. Our in vivo experiments using immunosuppressed mice showed that MI-1544, applied twice daily s.c., inhibited the growth of oestrogensensitive and -insensitive xenografts by 65% and 30% respectively. This effect was potentiated (70%) in both types of xenografts by the presence of the polymeric carrier in the conjugate; however, the carrier by itself did not cause tumour growth inhibition. The polymeric polypeptide carrier is supposed to increase the stability of the GnRH antagonist and to prevent the rapid excretion of the covalently bound peptide molecule. The antagonist and its conjugate may have various direct and indirect effects on breast cancer cells and, as a consequence, the new GnRH antagonist conjugates are suitable for treating an extended range of breast cancers.